Methods and compositions for pathogen detection in plants

ABSTRACT

The technology relates in part to methods and compositions for detecting one or more pathogens in plants. In some aspects, the technology relates to methods and compositions for detecting hops latent viroid in plants. In some aspects, the technology relates to methods and compositions for detecting hops latent viroid in  cannabis  plants. In some aspects, the technology relates to methods and compositions for classifying a hops latent viroid genotype. In certain aspects, the technology relates to methods and compositions for determining the presence, absence and/or amount of one or more pathogens in plants, either independently or simultaneously. In aspects, the pathogen is a virus. In some aspects, the virus is selected from among one or more of hops latent viroid, beet curly top virus and alfalfa mosaic virus.

RELATED PATENT APPLICATION

This patent application claims priority to U.S. Provisional PatentApplication No. 63/032,155 filed on May 29, 2020, entitled METHODS ANDCOMPOSITIONS FOR PATHOGEN DETECTION IN PLANTS, naming ChristopherStephen PAULI et al. as inventors, and designated by Attorney Docket No.FRB-1003-PV. The entire content of the foregoing patent application isincorporated herein by reference for all purposes.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jul. 19, 2021, isnamed FRB-1003-UTt_SL.txt and is 42,885 bytes in size.

FIELD

The technology relates in part to methods and compositions for detectingone or more pathogens in plants. In some aspects, the technology relatesto methods and compositions for detecting hops latent viroid in plants.In some aspects, the technology relates to methods and compositions fordetecting hops latent viroid in cannabis plants. In some aspects, thetechnology relates to methods and compositions for classifying a hopslatent viroid genotype. In certain aspects, the technology relates tomethods and compositions for determining the presence, absence and/oramount of one or more pathogens in plants, either independently orsimultaneously. In aspects, the pathogen is a virus or viroid. In someaspects, the virus or viroid is selected from among one or more of HopsLatent Viroid (HpLVd), Beet Curly Top Virus (BCTV) and Alfalfa MosaicVirus (AMV).

BACKGROUND

Cannabis is a genus of flowering plants that includes at least threespecies, Cannabis sativa, Cannabis indica, and Cannabis ruderalis, asdetermined by plant phenotypes and secondary metabolite profiles(chemotype). Both marijuana and hemp plants are in this genus andproduce a unique family of terpeno-phenolic compounds calledcannabinoids. The cannabinoids typically produced in greatest abundanceare cannabidiol (CBD) and Δ9-tetrahydrocannabinol (THC). CBD and THChave been shown to have different physiological effects when ingested.Cannabis is used to produce hemp fiber and hemp oil, for medicinalpurposes, and as a recreational drug. Hemp cultivars of cannabis arebred to produce minimal levels of THC, while marijuana cultivars arebred to produce higher levels of THC. CBD has been shown to have anumber of medically useful effects such as anti-inflammatory,anti-convulsant, antioxidant, antiemetic, anxiolytic and antipsychoticeffects, and THC is psychoactive. In general, the maximum THC content ofhemp is 0.3% and any cannabis with a THC content of greater than 0.3% isconsidered to be marijuana.

Cannabis plants can be susceptible to infection by pathogens. Pathogensmay include viruses, viroids, bacteria, fungi, nematodes, and/or anyorganisms that can cause disease in plants. Certain pathogens can reducethe quality and/or productivity of plants, and in certain instances,pathogens can cause plant death. Pathogens can be introduced and spreadto host plants in a variety of ways. For example, bacterial and fungalspores can be transmitted by wind, rain, and/or soil. Certain pathogenscan be spread through insects, transplants, infected seeds, irrigationwater contaminated equipment, and humans.

One pathogen capable of infecting Cannabis plants is the hops latentviroid (HpLVd). Symptoms of a hops latent viroid infection may includereduction or lack of oil, small heads, misshapen leaves, leaves that areyellowish in color, brittle stems, an outwardly horizontal plantstructure, and reduced flower mass and trichomes, although some plantsinfected with hops latent viroid or a hops latent viroid variant may beasymptomatic. Other pathogens with similar deleterious effects includeviruses such as Beet Curly Top Virus (BCTV) and Alfalfa Mosaic Virus(AMV). Given the potentially detrimental effects of hops latent viroidinfection and viruses such as BCTV and AMV in Cannabis plants, there isa need for accurate diagnostics of hops latent viroid and/or otherpathogenic infection and for an assessment of the relationship betweenhops latent viroid or other pathogenic variants and presentation ofsymptoms.

SUMMARY

Provided herein are diagnostics for detecting presence, absence and/oramount of pathogens in plant cultivars. In certain aspects, provided areaccurate diagnostics for HpLVd infection and for an assessment of therelationship between hops latent viroid variants and presentation ofsymptoms. Such diagnostics are useful given the potentially detrimentaleffects of hops latent viroid infection in plant cultivars (e.g.,Cannabis plant cultivars).

Provided in certain aspects are diagnostics that specifically andreproducibly identify more than one pathogen in plant cultivars,independently or simultaneously, e.g., in multiplexed methods. Suchdiagnostics are useful given the plethora of pathogens that can infectplant cultivars (e.g., Cannabis plant cultivars), including other plantviruses such as AMV and BCTV.

Provided herein, in some aspects, are methods for analyzing nucleic acidfrom a plant sample, comprising contacting nucleic acid of a plantsample with one or more polynucleotide primer pairs under amplificationconditions, thereby generating one or more amplification products; andanalyzing the amplification products; where the majority or all of theone or more polynucleotide primer pairs hybridize to subsequences of SEQID NO:1 if present in the nucleic acid of the plant sample under theamplification conditions; the subsequences of SEQ ID NO:1 to which themajority or all of the polynucleotide primers hybridize under theamplification conditions contain no variant nucleotide position; andeach subsequence of SEQ ID NO:1 between the subsequences to which theone or more primer pairs hybridize contain one or more variantnucleotide positions.

Also provided herein, in some aspects, are methods for generatingnucleic acid amplification products from a plant sample, comprisingcontacting nucleic acid of a plant sample with one or morepolynucleotide primer pairs under amplification conditions, therebygenerating one or more amplification products, where the majority or allof the one or more polynucleotide primer pairs hybridize to subsequencesof SEQ ID NO:1 if present in the nucleic acid of the plant sample underthe amplification conditions; the subsequences of SEQ ID NO:1 to whichthe majority or all of the polynucleotide primers hybridize under theamplification conditions contain no variant nucleotide position; andeach subsequence of SEQ ID NO:1 between the subsequences to which theone or more primer pairs hybridize contain one or more variantnucleotide positions.

Also provided herein, in some aspects, are methods for analyzing nucleicacid from a plant sample, comprising a) contacting nucleic acid of aplant sample with a first set of polynucleotide primers underamplification conditions, thereby generating a first set ofamplification products, where i) the majority or all of the primers inthe first set of polynucleotide primers hybridize to subsequences of SEQID NO:1 if present in the nucleic acid of the plant sample under theamplification conditions, ii) the subsequences of SEQ ID NO:1 to whichthe majority or all of the primers in the first set of polynucleotideprimers hybridize under the amplification conditions contain no variantnucleotide position, and iii) each subsequence of SEQ ID NO:1 betweenthe subsequences to which the primers in the first set of polynucleotideprimers hybridize contain one or more variant nucleotide positions; b)contacting the nucleic acid of the plant sample with a second set ofpolynucleotide primers under the amplification conditions, therebygenerating a second set of amplification products, where i) the majorityor all of the primers in the second set of polynucleotide primershybridize to subsequences of SEQ ID NO:1 if present in the nucleic acidof the plant sample under the amplification conditions, and ii) thesubsequences of SEQ ID NO:1 to which the majority or all of the primersin the second set of polynucleotide primers hybridize under theamplification conditions contain one or more variant nucleotidepositions; and c) analyzing the first and second sets of amplificationproducts.

Also provided herein, in some aspects, are methods for generatingnucleic acid amplification products from a plant sample, comprising a)contacting nucleic acid of a plant sample with a first set ofpolynucleotide primers under amplification conditions, therebygenerating a first set of amplification products, where i) the majorityor all of the primers in the first set of polynucleotide primershybridize to subsequences of SEQ ID NO:1 if present in the nucleic acidof the plant sample under the amplification conditions, ii) thesubsequences of SEQ ID NO:1 to which the majority or all of the primersin the first set of polynucleotide primers hybridize under theamplification conditions contain no variant nucleotide position, andiii) each subsequence of SEQ ID NO:1 between the subsequences to whichthe primers in the first set of polynucleotide primers hybridize containone or more variant nucleotide positions; and b) contacting the nucleicacid of the plant sample with a second set of polynucleotide primersunder the amplification conditions, thereby generating a second set ofamplification products, where i) the majority or all of the primers inthe second set of polynucleotide primers hybridize to subsequences ofSEQ ID NO:1 if present in the nucleic acid of the plant sample under theamplification conditions, and ii) the subsequences of SEQ ID NO:1 towhich the majority or all of the primers in the second set ofpolynucleotide primers hybridize under the amplification conditionscontain one or more variant nucleotide positions.

Also provided herein, in some aspects, are methods for analyzing nucleicacid from a plant sample, comprising contacting nucleic acid of a plantsample with a plurality of polynucleotide primer pairs underamplification conditions, thereby preparing a mixture; and analyzingnucleic acid of the mixture; where the majority or all of thepolynucleotide primer pairs hybridize to subsequences of SEQ ID NO:1 ifpresent in the nucleic acid of the plant sample under the amplificationconditions; the subsequences of SEQ ID NO:1 to which the majority or allof the polynucleotide primers hybridize under the amplificationconditions contain no variant nucleotide position or one variantnucleotide position; and each subsequence of SEQ ID NO:1 between thesubsequences to which the primer pairs hybridize contain two or morevariant nucleotide positions.

Also provided herein, in some aspects, are methods for preparing anucleic acid mixture comprising contacting nucleic acid of a plantsample with a plurality of polynucleotide primer pairs underamplification conditions, thereby preparing a mixture, where themajority or all of the polynucleotide primer pairs hybridize tosubsequences of SEQ ID NO:1 if present in the nucleic acid of the plantsample under the amplification conditions; the subsequences of SEQ IDNO:1 to which the majority or all of the polynucleotide primershybridize under the amplification conditions contain no variantnucleotide position or one variant nucleotide position; and eachsubsequence of SEQ ID NO:1 between the subsequences to which the primerpairs hybridize contain two or more variant nucleotide positions.

Also provided herein, in some aspects, are compositions comprising oneor more polynucleotide primer pairs where each polynucleotide of the oneor more primer pairs is identical, or substantially identical, to asubsequence of SEQ ID NO:1, or complement thereof; each subsequence ofSEQ ID NO:1, or complement thereof, to which each polynucleotide isidentical, or substantially identical, contains no variant nucleotideposition; and each target sequence of SEQ ID NO:1 between thesubsequences, or complements thereof, to which the polynucleotides ofthe one or more primer pairs are identical, or substantially identical,comprises one or more variant nucleotide positions.

Also provided herein, in some aspects, are compositions comprising a) afirst set of polynucleotide primers where i) each polynucleotide of thea first set of polynucleotide primers is identical, or substantiallyidentical, to a subsequence of SEQ ID NO:1, or complement thereof, ii)each subsequence of SEQ ID NO:1, or complement thereof, to which eachpolynucleotide is identical, or substantially identical, contains novariant nucleotide position, and iii) each target sequence of SEQ IDNO:1 between the subsequences, or complements thereof, to which thepolynucleotides of the first set of polynucleotide primers areidentical, or substantially identical, comprises one or more variantnucleotide positions; and b) a second set of polynucleotide primerswhere i) each polynucleotide of the second set of polynucleotide primersis identical, or substantially identical, to a subsequence of SEQ IDNO:1, or complement thereof, and ii) each subsequence of SEQ ID NO:1, orcomplement thereof, to which each polynucleotide is identical, orsubstantially identical, contains one or more variant nucleotidepositions.

Also provided herein, in some aspects, is a method for determining thepresence, absence and/or amount of a pathogen in a plant cultivar,comprising: (a) obtaining a nucleic acid sample from the plant cultivar;(b) contacting the nucleic acid sample with at least one polynucleotideprimer pair under amplification conditions and amplifying the sample,thereby preparing an amplified nucleic acid mixture, wherein, if thepathogen is present, the polynucleotide primer pair is capable ofspecifically hybridizing to and amplifying a subsequence of the nucleicacid of the pathogen, or to a complement thereof, wherein thesubsequence of the nucleic acid of the pathogen, or the complementthereof, is non-identical (i.e., not identical) to any subsequence ofthe nucleic acid of the plant genome, or to any complement thereof; and(c) determining the presence, absence and/or amount of at least oneamplicon that is 300 base pairs or less and is an amplification productof the polynucleotide primer pair in the amplified nucleic acid mixtureof (b), thereby determining the presence, absence and/or amount of apathogen in the plant cultivar.

In certain aspects, provided herein is a method of preparing a nucleicacid mixture from a plant cultivar, comprising:

-   -   (a) obtaining a nucleic acid sample from the plant cultivar; and    -   (b) preparing an amplified nucleic acid mixture by contacting        the nucleic acid sample with at least one polynucleotide primer        pair under amplification conditions and amplifying the sample,        wherein, if the pathogen is present, the polynucleotide primer        pair is capable of specifically hybridizing to and amplifying a        subsequence of the nucleic acid of the pathogen, or to a        complement thereof, wherein the subsequence of the nucleic acid        of the pathogen, or the complement thereof, is non-identical to        any subsequence of the nucleic acid of the plant genome, or to        any complement thereof. In aspects, the method further        comprises, determining the presence, absence and/or amount of at        least one amplicon that is 300 base pairs or less and is an        amplification product of the polynucleotide primer pair in the        amplified nucleic acid mixture of (b), thereby determining the        presence, absence and/or amount of a pathogen in the plant        cultivar.

In aspects, in any of the methods provided herein, the subsequence ofthe nucleic acid of the pathogen, or the complement thereof, is in aregion of overlap between two genes in the genome of the pathogen. Incertain aspects, the pathogen is a virus or viroid. In aspects, thevirus or viroid is selected from among Hops Latent Viroid (HpLVd),Alfalfa Mosaic Virus (AMV), Beet Curly Top Virus (BCTV), Hemp StreakVirus (HSV), Hemp Mosaic Virus (HMV), Tomato spotted wilt virus (TSWV),Sunn-Hemp Mosaic Virus (SHMV), Arabis Mosaic Virus (ArMV), CucumberMosaic Virus (CMV), Lettuce Chlorosis Virus (LCV), Tobacco RingspotVirus (TRSV), Tomato Ringspot Virus (TomRSV), and Tobacco Streak Virus(TSV), Cannabis Cryptic Virus (CCV), Potato Spindle Tubular Viroid(PSTV), Coconut cadang cadang viroid (CCCV), Apple scar skin viroid(ASSV), Avocado sunblotch viroid (ASBV), Tobacco streak virus (TSV),Tomato mosaic virus (ToMV), Euonymous Ringspot Virus (ERSV), Elm MosaicVirus (EMV), and Hops Stunting Virus (HpSV).

Also provided herein, in certain aspects, are multiplexed methods ofdetermining the presence, absence and/or amount of one or more pathogensin one or more plant cultivars. In certain aspects, the multiplexedmethod comprises one or more of:

-   -   (1) determining the presence, absence and/or amount of more than        one non-overlapping amplicon of a pathogen that may have        infected a plant cultivar;    -   (2) determining the presence, absence and/or amount of more than        one pathogen that may have infected a plant cultivar by        determining the presence, absence and/or amount of one or more        amplicons of each pathogen;    -   (3) determining the presence, absence and/or amount of one or        more pathogens in a plurality of plant cultivars.

In aspects, the multiplexed methods provided herein are for determiningthe presence, absence and/or amount of one or more of the followingpathogens in a plant cultivar: In aspects, the virus is selected fromamong one or more of Hops Latent Viroid (HpLVd), Alfalfa Mosaic Virus(AMV), Beet Curly Top Virus (BCTV), Hemp Streak Virus (HSV), Hemp MosaicVirus (HMV), Tomato spotted wilt virus (TSWV), Sunn-Hemp Mosaic Virus(SHMV), Arabis Mosaic Virus (ArMV), Cucumber Mosaic Virus (CMV), LettuceChlorosis Virus (LCV), Tobacco Ringspot Virus (TRSV), Tomato RingspotVirus (TomRSV), and Tobacco Streak Virus (TSV), Cannabis Cryptic Virus(CCV), Potato Spindle Tubular Viroid (PSTV), Coconut cadang cadangviroid (CCCV), Apple scar skin viroid (ASSV), Avocado sunblotch viroid(ASBV), Tobacco streak virus (TSV), Tomato mosaic virus (ToMV),Euonymous Ringspot Virus (ERSV), Elm Mosaic Virus (EMV), and HopsStunting Virus (HpSV). In aspects, the virus is selected from among oneor more of Hops Latent Viroid (HpLVd), Alfalfa Mosaic Virus (AMV), BeetCurly Top Virus (BCTV).

In any of the methods provided herein, in certain aspects, determiningthe presence, absence and/or amount of one or more amplicons of a plantpathogen is by quantitative PCR (qPCR), or quantitative RT-PCR(RT-qPCR). In aspects, the one or more amplicons are quantified using apolynucleotide probe sequence. In certain aspects, an amplicon of atleast one pathogen is quantified with more than one polynucleotide probesequence, wherein the polynucleotide probe sequences hybridize tonon-overlapping regions of the subsequence of the pathogen that isamplified to generate the amplicon.

In aspects, if the presence, absence and/or amount of one pathogen inthe plant cultivar is to be determined, more than one amplicon can beobtained by amplifying more than one subsequence of the nucleic acid ofthe pathogen, or complements thereof, using more than one polynucleotideprimer pair, and determining the presence, absence and/or amount of thepathogen by determining the presence, absence and/or amount of at leasttwo amplicons that are 300 base pairs or less and are amplificationproducts of the more than one polynucleotide primer pair in theamplified nucleic acid mixture, thereby determining the presence,absence and/or amount of a pathogen in the plant cultivar. In certainaspects, if the presence, absence and/or amount of a plurality ofpathogens in the plant cultivar is to be determined, more than oneamplicon can be obtained by amplifying more than one subsequence of thenucleic acid of more than one of the plurality of pathogens, orcomplements thereof, using more than one polynucleotide primer pair foreach of the more than one pathogens, and determining the presence,absence and/or amount of the more than one pathogens by determining thepresence, absence and/or amount of at least two amplicons for eachpathogen that are 300 base pairs or less and are amplification productsof the more than one polynucleotide primer pair in each of the more thanone pathogens of the amplified nucleic acid mixture of, therebydetermining the presence, absence and/or amount of the more than onepathogens in the plant cultivar.

In aspects, determining the presence, absence and/or amount of ampliconsobtained by a polynucleotide primer pair specifically hybridizing to andamplifying one or more subsequences of one or more plant pathogens is byRT-qPCR or qPCR, and the one or more amplicons, if present, arequantified using polynucleotide probes. A Cq value can be determined foreach polynucleotide probe, whereby, if the Cq value is above a thresholdvalue, the presence and/or amount of an amplicon is determined, therebydetermining the presence and/or amount of a pathogen in a plant cultivarand if the Cq value is below a threshold value, the absence of anamplicon is determined, thereby determining the absence of a pathogen ina plant cultivar. In certain aspects, more than one non-overlappingprobe is used to quantify an amplicon obtained by a polynucleotideprimer pair specifically hybridizing to and amplifying a subsequence ofa plant pathogen and, if the Cq value obtained with a firstpolynucleotide probe sequence is significantly different than the Cqvalue obtained with any of the other non-overlapping polynucleotideprobe sequences, a variant in the genotype of the pathogen is identifiedand, if the Cq value obtained with a first polynucleotide probe sequenceis similar to the Cq values obtained with any of the othernon-overlapping polynucleotide probe sequences, the genotype of thepathogen is identified as not comprising a variant genotype of thepathogen. In aspects, the presence or absence of a variant in thegenotype of the pathogen is correlated to the infectivity of thepathogen. In aspects, more than one non-overlapping subsequence of apathogen is amplified to obtain and quantify more than one amplicon and,based on the relative Cq values for each amplicon, the presence orabsence of a variant in the genotype of the pathogen is identified. Inaspects, the presence or absence of a variant in the genotype of thepathogen is correlated to the infectivity of the pathogen. In someaspects, the presence or absence of a variant in the genotype of thepathogen is correlated to resistance or susceptibility of the plant toinfection by the pathogen comprising the genotype or a variant thereof.As used herein, Cq, Cp and Ct values are measures of the same cyclethreshold value using different software, e.g., Thermofisher Scientific,Waltham, Mass. (Cq), Roche Diagostics, Indianapolis, Ind. (Cp) andBio-Rad Diagnostics, Hercules, Calif. (Ct).

In aspects of the methods provided herein, a positive control ampliconis generated using a polynucleotide primer pair that is capable ofspecifically hybridizing to and amplifying a subsequence of the nucleicacid of the plant genome, or to a complement thereof, wherein thesubsequence of the nucleic acid of the plant genome, or the complementthereof, is non-identical to any subsequence of the nucleic acid of thepathogen, or to any complement thereof; and determining the presence,absence and/or amount of at least one amplicon that is an amplificationproduct of the polynucleotide primer pair that is capable ofspecifically hybridizing to and amplifying a subsequence of the nucleicacid of the plant genome, thereby determining whether the amplificationconditions are effective for generating amplicons. In aspects, thesubsequence of the nucleic acid of the plant genome comprises all orpart of a gene selected from among 26S rRNA, beta-tubulin, ATP Synthase,an rRNA subunit, glyceraldehyde-3-phosphate dehydrogenase,Ubiquitin-conjugating enzyme E2, eukaryotic transcription factors,eukaryotic initiation factor 1 and beta-actin.

In any of the methods provided herein, in aspects, the subsequence ofthe nucleic acid of the pathogen, or the complement thereof, comprisesall or a portion of at least one gene that is conserved among species ofthat pathogen. In aspects, the at least one gene that is conserved amongspecies of the pathogen is selected from among RNA-3 coat protein,SS-ds-DNA Regulator protein, Movement Protein, Pathogenesis EnhancerProtein, Rolling Circle Replication Protein, Cell Cycle RegulatorProtein and Replication Enhancer Protein.

In aspects, the pathogen is Alfalfa Mosaic Virus (AMV). In certainaspects, the subsequence of the nucleic acid of the pathogen to whichthe polynucleotide primer pair is capable of hybridizing comprises SEQID NO:91, or a portion of SEQ ID NO:91, or a complement of SEQ ID NO:91,or a portion of the complement of SEQ ID NO:91.

In certain aspects, the pathogen is HpLVd. In aspects, the subsequenceof the nucleic acid of the pathogen to which the polynucleotide primerpair is capable of hybridizing comprises SEQ ID NO:1, or a portion ofSEQ ID NO:1, or a complement of SEQ ID NO:1, or a portion of thecomplement of SEQ ID NO:1.

In certain aspects, the pathogen is BCTV. In aspects, the subsequence ofthe nucleic acid of the pathogen to which the polynucleotide primer pairis capable of hybridizing is selected from among SEQ ID NOS:110, 112,114, 116, 118 or 120, or a portion of SEQ ID NOS:110, 112, 114, 116, 118or 120, or a complement of SEQ ID NOS:110, 112, 114, 116, 118 or 120, ora portion of the complement of SEQ ID NOS:110, 112, 114, 116, 118 or120, or to regions of overlap that spans any two of SEQ ID NOS:110, 112,114, 116, 118 or 120 in the genome of the pathogen.

In aspects of the methods provided herein, the presence, absence and/oramount of more than one pathogen selected from among Hops Latent Viroid(HpLVd), Alfalfa Mosaic Virus (AMV) and Beet Curly Top Virus (BCTV) isdetermined simultaneously. In certain aspects, the plant cultivar is aCannabis cultivar. In aspects, the method is a multiplexed method inwhich the presence, absence and/or amount of one or more pathogens isdetermined in a plurality of plant cultivars. In aspects, one, aportion, or all of the plant cultivars of the plurality is/are Cannabiscultivars.

Any of the methods provided herein can, in certain aspects, be performedon a solid support. In aspects, the solid support comprises a bead,column, capillary, disk, filter, dipstick, membrane, wafer, comb, pin ora chip.

Also provided herein, in aspects, is a method of preparing apolynucleotide primer pair for specifically hybridizing to andamplifying nucleic acid of a plant pathogen, comprising:

-   -   (a) Identifying a polynucleotide primer pair that is capable of        specifically hybridizing to and amplifying a polynucleotide        comprising a subsequence of the nucleic acid of a plant        pathogen, or a complement thereof, wherein the plant is capable        of being infected by the pathogen and the subsequence of the        nucleic acid of the pathogen, or the complement thereof, is        non-identical to any subsequence of the nucleic acid of the        plant genome, or to any complement thereof;    -   (b) identifying whether the subsequence of the nucleic acid of        the pathogen is conserved among species of the pathogen; and    -   (c) if the subsequence of the nucleic acid of the pathogen is        conserved among species of the pathogen, preparing the        polynucleotide primer pair.

Also provided, in certain aspects, are compositions comprising one ormore polynucleotide primer pairs prepared by the methods providedherein. Also provided herein, in certain aspects, are compositionscomprising one or more polynucleotide primer pairs used in the methodsprovided herein for specifically hybridizing to and amplifying nucleicacid of a plant pathogen and, optionally, one or more polynucleotideprobes provided herein for quantifying one or more amplicons generatedusing the one or more polynucleotide primer pairs. In aspects, providedherein are kits comprising one or more of the compositions providedherein, and instructions for use.

Also provided herein, in aspects, are solid supports, comprising:

-   -   single-stranded nucleic acid from a plant cultivar; and    -   one or more polynucleotide primer pairs used in the methods        provided herein or one or more polynucleotide primer pairs        prepared by the methods provided herein for specifically        hybridizing to and amplifying nucleic acid of a plant pathogen.        In aspects, the solid support comprises a bead, column,        capillary, disk, filter, dipstick, membrane, wafer, comb, pin or        a chip.

Certain embodiments are described further in the following description,examples, claims and drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

The drawings illustrate certain embodiments of the technology and arenot limiting. For clarity and ease of illustration, the drawings are notmade to scale and, in some instances, various aspects may be shownexaggerated or enlarged to facilitate an understanding of particularembodiments.

FIG. 1 shows results of an optimization of general assay components fora hops latent viroid RT-qPCR method for primer pair A-G (A-fwd, B-rev)and probe p1. MM, master mix.

FIG. 2 shows results of an RT-qPCR analysis of primer/probe combinationsfor primer pairs A-D (A-fwd, D-rev), A-E (A-fwd, E-rev), and A-F (A-fwd,F-rev) and probes p1-p5 under optimized reaction condition 7.

FIG. 3 shows results of an RT-qPCR analysis of primer/probe combinationsfor primer pairs A-G (A-fwd, G-rev), B-D (B-fwd, D-rev), and B-E (B-fwd,E-rev) and probes p1-p5 under optimized reaction condition 7.

FIG. 4 shows results of an RT-qPCR analysis of primer/probe combinationsfor primer pairs B-F (B-fwd, F-rev) and B-G (B-fwd, G-rev) and probesp1-p5 under optimized reaction condition 7.

FIG. 5 shows results of an RT-qPCR analysis of the primer pair A-G(A-fwd, G-rev) with probe p1 and primer pair F-D (F-fwd, D-rev) withprobe p3 performed on known positive and negative test samples. Thearrows point to background and/or late cycle amplification.

FIG. 6 shows results of an RT-qPCR analysis of unknown test samplesperformed using primer pair A-G (A-fwd, G-rev) with probe p1, and primerpair B-G (B-fwd, G-rev) with probe p5 using Gel CZ1 as a positivecontrol and no template as a negative control. The arrow points tobackground and/or late cycle amplification.

FIG. 7 shows results of an RT-qPCR analysis of genomic DNA and testRNA/cDNA samples was performed using primer pair A-G (A-fwd, G-rev) withprobe p1, and primer pair B-G (B-fwd, G-rev) with probe p5 using Gel CZ1as a positive control and no template as a negative control.

FIG. 8 shows results of melt curve genotyping analysis performed usingprimer pairs A-A (A-fwd, A-rev), A-B (A-fwd, B-rev), and A-C (A-fwd,C-rev).

FIG. 9 shows an example illustration of LAMP primers.

FIG. 10 shows an example illustration of a LAMP assay.

FIGS. 11A-11B depict a validation analysis of multiplexed determinationof the presence, absence and/or amount of pathogen in HpLVd-positiveCannabis cultivar samples spiked with AMV. FIG. 11A is a Table listingthe Cq values for the reaction conditions tested, and FIG. 11B shows theamplification plots for various primer and probe sets as indicated onthe top left of each plot.

FIGS. 12A-12B depict multiplexed RT-qPCR for determining the presence,absence and/or amount of HpLVd, AMV and BCTV in Cannabis cultivars. FIG.12A is a Table listing the Cq values for the reaction conditions tested,and FIG. 12B shows the amplification plots for samples and targets asindicated.

FIGS. 13A-13B depict the reproducibility of multiplexed RT-qPCR fordetermining the presence, absence and/or amount of HpLVd and BCTV in RNAfrom pooled leaf samples of Cannabis cultivars. FIG. 13A is a Tablelisting the Cq values for the reaction conditions tested, and FIG. 13Bshows the amplification plots for samples and targets as indicated.

FIGS. 14A-14C depict the sensitivity of multiplexed RT-qPCR as measuredby a standard curve. FIG. 14A is a Table listing the Cq values for thereaction conditions tested, and FIG. 14B shows the amplification plotsfor samples and targets as indicated. FIG. 14C depicts standard curvesfor the detection of HpLVd, AMV and BCTV.

FIG. 15 depicts the sensitivity and specificity for detection of HPLVd,BCTV, and AMV in a RT-qPCR assay.

FIG. 16 depicts validation of a High throughput RT-qPCR Method fordetection of pathogens in a plant.

FIG. 17 depicts a High throughput LAMP Method for detection of pathogensin a plant.

DETAILED DESCRIPTION

Provided herein are methods and compositions for determining thepresence, absence and/or amount of a pathogen in a plant cultivar, whichinclude: (a) obtaining a nucleic acid sample from the plant cultivar;(b) contacting the nucleic acid sample with at least one polynucleotideprimer pair under amplification conditions and amplifying the sample,thereby preparing an amplified nucleic acid mixture, wherein, if thepathogen is present, the polynucleotide primer pair is capable ofspecifically hybridizing to and amplifying a subsequence of the nucleicacid of the pathogen, or to a complement thereof, wherein thesubsequence of the nucleic acid of the pathogen, or the complementthereof, is not identical (i.e., non-identical) to any subsequence ofthe nucleic acid of the plant genome, or to any complement thereof; and(c) determining the presence, absence and/or amount of at least oneamplicon that is 300 base pairs or less and is an amplification productof the polynucleotide primer pair in the amplified nucleic acid mixtureof (b), thereby determining the presence, absence and/or amount of apathogen in the plant cultivar.

In certain embodiments, the plant is a member of the Rosidae subclass.In embodiments, the plant is a Cannabis plant. Any type of Cannabisplant can be analyzed according to the methods provided hereinincluding, but not limited to, Type 1 (THC-dominant), Type 2 (Mixedratio—CBD & THC), Type 3 (CBD-dominant), Type 4 (CBG-dominant) and Type5 (Varin-dominant).

The methods and compositions provided herein can, in certainembodiments, be used in a multiplexed format to analyze one or more of:(1) more than one pathogen in a single plant cultivar; (2) more than onesubsequence of a single pathogen; (3) a single subsequence of a pathogenquantified using more than one polynucleotide probe for quantificationof the amplicon obtained by a polynucleotide primer pair that is capableof specifically hybridizing to and amplifying a subsequence of thenucleic acid of the pathogen; and/or (4) one or more pathogens in aplurality of plant cultivars.

The polynucleotide primer pair for specifically hybridizing to andamplifying a subsequence of the nucleic acid of the pathogen, or to acomplement thereof, binds to a subsequence of the nucleic acid of thepathogen, or the complement thereof, that is non-identical to anysubsequence of the nucleic acid of the plant genome, or to anycomplement thereof.

In the methods and compositions provided herein, in embodiments, thepolynucleotide primer pairs for specifically hybridizing to andamplifying a subsequence of the nucleic acid of the pathogen aredesigned to amplify a subsequence that is non-identical to anysubsequence of the nucleic acid of the plant genome, thereby permittingspecific detection of the plant pathogen and avoiding non-specificdetection of sequences of the plant nucleic acid. In certainembodiments, the subsequence of the nucleic acid of the pathogen is in acoding region, thereby permitting the detection of pathogens that areactively expressing proteins and/or are replicating in the plant (e.g.,detecting RNA or cDNA of a plant virus, rather than latent virus). Inembodiments, the subsequence of the nucleic acid of the pathogen is in aregion of overlap between the coding sequences of more than one proteinexpressed by the pathogen, thereby permitting better confirmation of theidentity of the pathogen. In certain embodiments, the identity and/orgenotypic variation in a pathogen can be determined by amplifying morethan one non-overlapping subsequence of the nucleic acid pathogen, usingmore than one polypeptide primer pair.

In embodiments, the amplicons generated by specific hybridization andamplification of such subsequences of the nucleic acid of the pathogencan be quantified, e.g., by qPCR or RT-qPCR, e.g., using polynucleotideprobes. In such quantification methods, the presence, absence and/oramount of an amplicon is determined by the threshold value of a signalor a parameter, such as a Cq (used interchangeably with Ct) value. Ingeneral, a value above (or that crosses) a threshold value indicatesthat an amplicon (and, therefore, the corresponding pathogen) ispresent, and a value at or below the threshold value indicates that theamplicon (and, therefore, the corresponding pathogen) is absent.Threshold values can be determined by methods known to those of skill inthe art, including, e.g., by obtaining a standard curve (see, e.g.,Example 6). The term “Cq” value (or “Ct” value), as used herein, refersto the number of cycles required for a signal, such as a fluorescentsignal obtained by labelling the primers and/or templates foramplification, to exceed the background signal (e.g., fluorescence).

In certain embodiments, an amplicon generated by amplifying asubsequence of the nucleic acid of a pathogen can be quantified usingmore than one non-overlapping polynucleotide probe, and differencesbetween the Cq values of the non-overlapping polynucleotide probes canprovide information regarding the presence or absence of genotypicvariation in the pathogen.

The pathogens can include viruses, viroids, bacteria, fungi, nematodes,and/or any organisms that can cause disease in plants. In certainembodiments, the pathogen is a virus. The virus can be a DNA virus or anRNA virus. In embodiments, the virus is selected from among Hops LatentViroid (HpLVd), Alfalfa Mosaic Virus (AMV), Beet Curly Top Virus (BCTV),Hemp Streak Virus (HSV), Hemp Mosaic Virus (HMV), Tomato spotted wiltvirus (TSWV), Sunn-Hemp Mosaic Virus (SHMV), Arabis Mosaic Virus (ArMV),Cucumber Mosaic Virus (CMV), Lettuce Chlorosis Virus (LCV), TobaccoRingspot Virus (TRSV), Tomato Ringspot Virus (TomRSV), and TobaccoStreak Virus (TSV), Cannabis Cryptic Virus (CCV), Potato Spindle TubularViroid (PSTV), Coconut cadang cadang viroid (CCCV), Apple scar skinviroid (ASSV), Avocado sunblotch viroid (ASBV), Tobacco streak virus(TSV), Tomato mosaic virus (ToMV), Euonymous Ringspot Virus (ERSV), ElmMosaic Virus (EMV), and Hops Stunting Virus (HpSV). In certainembodiments, the presence, absence and/or amount of more than onepathogen is determined simultaneously in one or more plant cultivars. Inembodiments, the virus is selected from among Hops Latent Viroid(HpLVd), Alfalfa Mosaic Virus (AMV) and Beet Curly Top Virus (BCTV).

Primer sequences and length may affect hybridization to target nucleicacid sequences. Depending on the degree of mismatch between the primerand target nucleic acid, low, medium or high stringency conditions maybe used to effect primer/target annealing. As used herein, the term“stringent conditions” refers to conditions for hybridization andwashing. Methods for hybridization reaction temperature conditionoptimization are known to those of skill in the art and may be found,for example, in Current Protocols in Molecular Biology, John Wiley &Sons, N.Y., 6.3.1-6.3.6 (1989); either aqueous or non-aqueous methodsare described in that reference and either can be used. Non-limitingexamples of stringent hybridization conditions are hybridization in 6×sodium chloride/sodium citrate (SSC) at about 45° C., followed by one ormore washes in 0.2×SSC, 0.1% SDS at 50° C. Another example of stringenthybridization conditions are hybridization in 6× sodium chloride/sodiumcitrate (SSC) at about 45° C., followed by one or more washes in0.2×SSC, 0.1% SDS at 55° C. A further example of stringent hybridizationconditions is hybridization in 6× sodium chloride/sodium citrate (SSC)at about 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at60° C. Often, stringent hybridization conditions are hybridization in 6×sodium chloride/sodium citrate (SSC) at about 45° C., followed by one ormore washes in 0.2×SSC, 0.1% SDS at 65° C. More often, stringencyconditions are 0.5M sodium phosphate, 7% SDS at 65° C., followed by oneor more washes at 0.2×SSC, 1% SDS at 65° C. Stringent hybridizationtemperatures can also be altered (i.e., lowered) with the addition ofcertain organic solvents, formamide for example. Organic solvents, likeformamide, reduce the thermal stability of double-strandedpolynucleotides, so that hybridization can be performed at lowertemperatures, while still maintaining stringent conditions and extendingthe useful life of nucleic acids that may be heat labile. As usedherein: stringency of hybridization in determining percentage mismatchare those conditions understood by those of skill in the art andtypically are substantially equivalent to the following: 1) highstringency: 0.1×SSPE, 0.1% SDS, 65° C.; 2) medium stringency: 0.2×SSPE,0.1% SDS, 50° C.; 3) low stringency: 1.0×SSPE, 0.1% SDS, 50° C. It isunderstood that equivalent stringencies may be achieved usingalternative buffers, salts and temperatures.

The terms “specifically hybridizes,” “specific hybridization” and thelike, as used herein, refers to conditions under which a polynucleotideprimer pair preferentially hybridizes to a particular subsequence, e.g.,of the nucleic acid of a pathogen, and hybridizes to a substantiallylesser degree, e.g., 5% or less, such as 5%, 4%, 3%, 2%, 1% or 0%, orbetween 0% to 1%, 2%, 3%, 4% or 5% or less, to any other subsequence ofthe nucleic acid of the pathogen, or to subsequences of the nucleic acidof any other pathogens, or to subsequences of the nucleic acid of aplant cultivar. In embodiments, the specific hybridization is underconditions of high stringency, or under conditions of medium stringency.

In embodiments of the methods and compositions provided herein, thepolynucleotide primer pairs specifically hybridize to and amplify asubsequence of a nucleic acid of a pathogen that is non-identical to oneor more of: (1) any of the other subsequences of the nucleic acid of thepathogen, or complements thereof; (2) subsequences of the nucleic acidof any other pathogens, or complements thereof; and (3) subsequences ofthe nucleic acid of the genome of the plant cultivar. A sequence that isnon-identical to another subsequence, or complement thereof, such asbeing non-identical to another subsequence of the plant genome, such asa Cannabis genome, generally refers to a sequence containing one or moremismatched nucleotides when compared to another subsequence ofequivalent length (e.g., identical length, a length that is about 95%,about 96%, about 97%, about 98%, about 99%, about 100%, about 101%,about 102%, about 103%, about 104% or about 105% of the length of thesubsequence to which it is compared, or a length that is longer orshorter by one nucleotide, two nucleotides, or three nucleotides thanthe subsequence to which it is compared) in the plant genome (e.g.,Cannabis genome, such as the CS10 Cannabis genome). In certainembodiments, the length of the sequence to which the subsequence ofequivalent length is compared is about 15 nucleotides to about 30nucleotides, or a length that is about 95%, about 96%, about 97%, about98%, about 99%, about 100%, about 101%, about 102%, about 103%, about104% or about 105% of a sequence of length between about 15 nucleotidesto about 30 nucleotides.

The polynucleotide primer pairs of the methods and compositions providedherein generally are between about 15 nucleotides to about 30nucleotides in length, generally about 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29 or 30 nucleotides in length, or about 18, 19, 20,21, 22, 23, 24, 25, 26 or 27 nucleotides in length. In some embodiments,the nucleic acid subsequence of the pathogen, to which a polynucleotideprimer pair specifically hybridizes and amplifies, comprises anon-identical sequence comprising at least one, two, three, four, five,six, seven, eight, nine or ten or more mismatches when compared to anyother subsequence of equivalent length (e.g., any subsequence ofequivalent length within the nucleic acid of the pathogen, orsubsequences of the nucleic acid of other pathogens, or subsequences ofthe nucleic acid of the plant genome). In embodiments, the nucleic acidsubsequence of the pathogen, to which a polynucleotide primer pairspecifically hybridizes and amplifies, is unique and comprises at leastone mismatch when compared to one or more of the following subsequences:(i) any other subsequence of equivalent length in the same pathogen, or(ii) any other subsequence of equivalent length in another pathogen(e.g., one or more other pathogens), or (iii) to any other subsequenceof equivalent length in the nucleic acid of the plant genome, or (iv) acombination of (i) and (ii), or (ii) and (iii), or (i) and (iii), or(i), (ii) and (iii). In certain embodiments, the nucleic acidsubsequence of the pathogen, to which a polynucleotide primer pairspecifically hybridizes and amplifies, is unique and includes at leastone mismatch when compared to any other subsequence of equivalent lengthin the nucleic acid of the plant genome.

The subsequence of the nucleic acid of the pathogen that is amplifiedusing the methods and compositions provided herein generally is about300 base pairs or less, generally of a size that permits specificdetection of the pathogen while substantially avoiding non-specificamplification of sequences of the plant genome and providing betterconsistency and reproducibility in melting characteristics of theamplicons. In embodiments, the size of the product that is amplified bythe prepared polynucleotide primer pair is between about 50 base pairsto about 300 base pairs, or about 300, 290, 280, 275, 270, 265, 260,255, 250, 245, 240, 235, 230, 225, 220, 215, 210, 205 or 200 base pairsor less. In embodiments, the size of the product that is amplified bythe polynucleotide primer pair is between about 40 base pairs to about200 base pairs, or between about 50 base pairs to about 150 base pairs.In some embodiments, the nucleic acid sequence of the amplicon isnon-identical to and comprises a sequence comprising at least one, two,three, four, five, six, seven, eight, nine or ten or more mismatcheswhen compared to any subsequence of equivalent length (e.g., anysubsequence of equivalent length within the nucleic acid of thepathogen, or subsequences of the nucleic acid of other pathogens, orsubsequences of the nucleic acid of the plant genome). In embodiments,the nucleic acid sequence of the amplicon is unique and comprises atleast one mismatch when compared to one or more of any othersubsequences of the pathogen, or to any other subsequences of any otherpathogens, or to any subsequence of the nucleic acid of the plantgenome. In certain embodiments, the nucleic acid sequence of theamplicon is unique and comprises at least one mismatch when compared toany subsequence of the nucleic acid of the plant genome.

The primers of the polynucleotide primer pairs of the methods andcompositions provided herein generally share a high degree of sequenceidentity to the subsequence, or complement thereof, to which theyspecifically hybridize and amplify. In some embodiments, eachpolynucleotide in each primer pair comprises a sequence that is at leastabout 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identicalto a subsequence, or complement thereof, to which it specificallyhybridizes and amplifies. For example, a polynucleotide primer pair thatspecifically hybridizes to a particular subsequence, e.g., of thenucleic acid of a pathogen, would hybridize to a substantially lesserdegree, e.g., 5% or less, such as 5%, 4%, 3%, 2%, 1% or 0%, or between0% to 1%, 2%, 3%, 4% or 5% or less, to any other non-identicalsubsequence of the nucleic acid of the pathogen, or to non-identicalsubsequences of the nucleic acid of any other pathogens, or tonon-identical subsequences of the nucleic acid of a plant cultivar.

Provided herein are methods and compositions for detecting the presence,absence and/or amount of pathogens, such as viruses, in a plant. Inembodiments, the pathogen is a virus or viroid selected from among HopsLatent Viroid (HpLVd), Alfalfa Mosaic Virus (AMV), Beet Curly Top Virus(BCTV), Hemp Streak Virus (HSV), Hemp Mosaic Virus (HMV), Tomato spottedwilt virus (TSWV), Sunn-Hemp Mosaic Virus (SHMV), Arabis Mosaic Virus(ArMV), Cucumber Mosaic Virus (CMV), Lettuce Chlorosis Virus (LCV),Tobacco Ringspot Virus (TRSV), Tomato Ringspot Virus (TomRSV), andTobacco Streak Virus (TSV), Cannabis Cryptic Virus (CCV), Potato SpindleTubular Viroid (PSTV), Coconut cadang cadang viroid (CCCV), Apple scarskin viroid (ASSV), Avocado sunblotch viroid (ASBV), Tobacco streakvirus (TSV), Tomato mosaic virus (ToMV), Euonymous Ringspot Virus(ERSV), Elm Mosaic Virus (EMV), and Hops Stunting Virus (HpSV). The term“virus,” as used herein, refers to an infective organism comprisingnucleic acid and protein, wherein the organism multiplies by infecting ahost organism, such as a plant or animal, that is different than thevirus. A “viroid,” as used herein, refers to an infective organismcomprising nucleic acid, generally without protein, and smaller than avirus. Viroids, like viruses, can multiply by infecting a host organismthat is different than the viroid. Both “virus” and “viroid,” as usedherein, are terms of art known to and understood by those of skill inthe art.

In certain embodiments, provided herein are methods for detecting thepresence, absence and/or amount of pathogens such as hops latent viroid(HpLVd), AMV and BCTV in a plant sample (e.g., a Cannabis plant sample).Also provided herein are methods and compositions for identifying anHpLVd, AMV or BCTV genotype in a plant sample. Also provided herein aremethods and compositions for classifying an HpLVd, AMV or BCTV genotype(e.g., associating one or more disease phenotypes in a plant (e.g., aCannabis plant) with a particular HpLVd genotype). Also provided hereinare methods and compositions for identifying an HpLVd, AMV or BCTVgenetic variation signature in a plant sample. Also provided herein aremethods and compositions for classifying an HpLVd, AMV or BCTV geneticvariation signature (e.g., associating one or more disease phenotypes ina plant sample (e.g., a Cannabis plant) with a particular HpLVd, AMV orBCTV genetic variation signature). As used herein, “a plant sample”refers to applying a method and/or composition described herein to oneplant sample in an assay, multiple plant samples each in a separateassay for each sample, multiple plant samples in a single assay, and anycombination of the foregoing.

In aspects of the methods and compositions provided herein, the genomeof the pathogen can be amplified and sequenced to identify a wild-typeor genotypic variant of the pathogen. In certain aspects, amplificationof the genome from a known pathogen-positive sample can serve as apositive control when performing the methods provided herein. Inaspects, the method is qPCR and in certain aspects, the method isRT-qPCR.

In embodiments, the methods provided herein are performed on cellulosepaper that includes chemicals that lyse the plant cells and denature theproteins while retaining the DNA for amplification and/or detection. Inembodiments, the cellulose paper is a FTA® card (Whatman).

Hops Latent Viroid (HpLVd), Alfalfa Mosaic Virus (AMV) and Beet CurlyTop Virus (BCTV)

Hops Latent Viroid (HpLVd)

Provided herein are methods for analyzing nucleic acid from a plantsample. In some embodiments, the analysis comprises detecting thepresence, absence or amount of a hops latent viroid (HpLVd) in the plantsample (e.g., a Cannabis plant sample). In some embodiments, theanalysis comprises determining one or more genotypes of a hops latentviroid (HpLVd). In some embodiments, the analysis comprises determininga genetic variation signature of a hops latent viroid (HpLVd). The hopslatent viroid (HpLVd), which also may be referred to as hop latentviroid, HLV, HLVd, or Putative Cannabis Infectious Agent (PCIA), wasfirst characterized as a pathogen in Humulus lupulus (hop) plants thatcan impact yield and secondary metabolite production. Such yield andmetabolite impacts generally are more pronounced in cannabis plants.HpLVd infections in cannabis may result in symptoms, or diseasephenotypes, such as loss of vigor, stunting, reduction in yield,reduction in potency, and/or changes in morphology (sometimescollectively referred to as “dudding”). Methods for treating plantsinfected with one or more pathogens (e.g., HpLVd) include thermotherapy(i.e., heat treatment), cold treatment, light treatment, plant growthregulator treatment (e.g., hormone treatment), and combinations thereof.One method for treating plants infected with HpLVd, or suspected ofbeing infected with HpLVd, is thermotherapy (i.e., heat treatment). Suchheat treatment typically reduces HpLVd levels, but may also lead to theaccumulation of sequence variability in the HpLVd genome. Sequencevariations induced by heat treatment may be referred to asthermomutants.

The complete sequence of the HpLVd genome (provided as GENBANK accessionno. NC_003611.1) is:

(SEQ ID NO: 1) CTGGGGAATACACTACGTGACTTACCTGTATGGTGGCAAGGGCTCGAAGAGGGATCCCCGGGGAAACCTACTCGAGCGAGGCGGAGATCGAGCGCCAGTTCGTGCGCGGCGACCTGAAGTTGCTTCGGCTTCTTCTTGTTCGCGTCCTGCGTGGAACGGCTCCTTCTTCACACCAGCCGGAGTTGGAAACTACCCGGTGGATACAACTCTTGAGCGCCGAGCTTTACCTGCAGAAGTTCACATAAAAAGT GCCCCT.

The reverse complement of the HpLVd genome also is contemplated herein:

(SEQ ID NO: 76) AGGGGCACTTTTTATGTGAACTTCTGCAGGTAAAGCTCGGCGCTCAAGAGTTGTATCCACCGGGTAGTTTCCAACTCCGGCTGGTGTGAAGAAGGAGCCGTTCCACGCAGGACGCGAACAAGAAGAAGCCGAAGCAACTTCAGGTCGCCGCGCACGAACTGGCGCTCGATCTCCGCCTCGCTCGAGTAGGTTTCCCCGGGGATCCCTCTTCGAGCCCTTGCCACCATACAGGTAAGTCACGTAGTGTATT CCCCAG.

Also provided herein are methods for detecting the presence or absenceof HpLVd variants and/or mutants (e.g., thermomutants). HpLVd variantsand/or mutants (e.g., thermomutants) may include any HpLVd having one ormore nucleotide substitutions, deletions, and/or insertions (e.g.,relative to SEQ ID NO:1). Non-limiting examples of HpLVd variants and/ormutants include Hop latent viroid isolate H2 (GENBANK accession no.EF613183.1), Hop latent viroid ‘thermomutant’ T75 (GEN BANK accessionno. AJ290409.1), Hop latent viroid isolate CV1 (GENBANK accession no.MK791751.1), Hop latent viroid isolate Y7 (GENBANK accession no.EF613192.1), Hop latent viroid isolate S5 (GENBANK accession no.EF613188.1), Hop latent viroid isolate K7 (GENBANK accession no.EF613185.1), Hop latent viroid ‘thermomutant’ T92 (GENBANK accession no.AJ290410.1), Hop latent viroid ‘thermomutant’ T59 (GENBANK accession no.AJ290407.1), Hop latent viroid ‘thermomutant’ T61 (GENBANK accession no.AJ290408.1), Hop latent viroid isolate A2 (GENBANK accession no.EF613181.1), Hop latent viroid ‘thermomutant’ T50 (GENBANK accession no.AJ290406.1), Hop latent viroid ‘thermomutant’ T40 (GENBANK accession no.AJ290405.1), Hop latent viroid ‘thermomutant’ T229 (GENBANK accessionno. AJ290412.1), Hop latent viroid ‘thermomutant’ T218 (GENBANKaccession no. AJ290411.1), Hop latent viroid ‘thermomutant’ T15 (GENBANK accession no. AJ290404.1), Hop latent viroid isolate GVdC_HLVd01(GENBANK accession no. KT600318.1), Hop latent viroid isolateGVdC_HLVd02 (GEN BANK accession no. KT600317.1), and Hop latent viroidsequence (GENBANK accession no. X07397.1). HpLVd variants and/or mutants(e.g., thermomutants) may include substitutions at one or more of thefollowing nucleotide positions (numbering relative to SEQ ID NO:1): 7,10, 12, 26, 27, 28, 29, 30, 33, 35, 43, 59, 121, 128, 134, 150, 157,162, 168, 169, 177, 200, 225, 229, 247, 248, and 253. Examples ofthermomutant substitutions include A to G at position 7 of SEQ ID NO:1,A to G at position 12 of SEQ ID NO:1, C to A at position 26 of SEQ IDNO:1, U to A at position 27 of SEQ ID NO:1, G to A at position 28 of SEQID NO:1, A to G at position 30 of SEQ ID NO:1, G to A at position 33 ofSEQ ID NO:1, G to A at position 35 of SEQ ID NO:1, C to U at position 43of SEQ ID NO:1, G to A at position 128 of SEQ ID NO:1, C to U atposition 150 of SEQ ID NO:1, C to U at position 157 of SEQ ID NO:1, C toA at position 162 of SEQ ID NO:1, U to Cat position 168 of SEQ ID NO:1,C to U at position 169 of SEQ ID NO:1, C to U at position 177 of SEQ IDNO:1, U to Cat position 229 of SEQ ID NO:1, A to G at position 247 ofSEQ ID NO:1, A to C at position 248 of SEQ ID NO:1, C to U at position253 of SEQ ID NO:1, and C to A at position 255 of SEQ ID NO:1. HpLVdvariants and/or mutants (e.g., thermomutants) may include one or morenucleotide insertions or deletions (e.g., deletion of U at position 225of SEQ ID NO:1).

In aspects of any of the methods provided herein, the entire 256 basepair genome of the HpLVd viroid can be amplified and sequenced toidentify a wild-type pathogen or genotypic variant thereof. In certainaspects, amplification of the HpLVd genome from a known positive samplecan be used as a positive control in the methods provided herein. Incertain aspects, the method is qPCR. In aspects, the method is RT-qPCR.An example of a primer set for amplifying the HpLVd genome is providedin the Table below:

Self Self 3′ Template GC comple- comple- Sequence (5′−>3′) strand LengthStart Stop Tm % mentarity mentarity Forward CTGGGGAATACACTACG Plus 22  1  22 59.24 50.00 4.00 2.00 primer TGACT (SEQ ID NO: 122) ReverseAGGGGCACTTTTTATGT Minus 22 256 235 58.16 40.91 3.00 1.00 primer GAACT(SEQ ID NO: 123) Product 256 length

Alfalfa Mosaic Virus (AMV)

In some embodiments, the analysis comprises detecting the presence,absence and/or amount of an Alfalfa Mosaic Virus (AMV) in the plantsample (e.g., a Cannabis plant sample). In some embodiments, theanalysis comprises determining one or more genotypes of an AMV. In someembodiments, the analysis comprises determining a genetic variationsignature of an AMV. Alfalfa mosaic virus (AMV), also known as LucerneMosaic Virus or Potato Calico Virus, is a phytopathogen that is foundworldwide and can damage a large variety of over 600 plant species,including commercially important crops such as Cannabis. The geneticmaterial of AMV consists of 3 linear single strands RNAs (RNA 1, RNA 2and RNA 3) and a subgenomic RNA (RNA 4) which is obtained bytranscription of the negative-sense strand of RNA 3. Symptoms caused byAMV infection vary from wilting, white flecks, malformation likedwarfing, ringspots, mottles, mosaics and necrosis depending on the *USstrain, host variety, stage of growth at infection and environmentalconditions. The virus can be detected in each part of the host plant,while the virions are mainly found in the cytoplasm of the infectedplant, as inclusion bodies.

Provided herein are methods and compositions for determining thepresence, absence and/or amount of AMV in a plant cultivar. In themethods and compositions provided herein, polynucleotide primer pairsare used to specifically hybridize to and amplify a subsequence of thenucleic acid of AMV, or a complement thereof, where the primer pairsand/or the subsequence are non-identical to any subsequence, orcomplement thereof, of equivalent length in the nucleic acid of theplant genome. In embodiments, the subsequence of the nucleic acid of AMVthat is amplified is a conserved sequence. In certain embodiments, thesubsequence of the nucleic acid of AMV, or a portion thereof, is in acoding region, or in a region of overlap between more than one gene ofthe nucleic acid of the AMV, or in a region of overlap between more thanone coding region of the nucleic acid of the AMV.

In certain embodiments, the subsequence of the nucleic acid of the AMVthat is amplified is a subsequence of RNA 3, having the sequence setforth below as SEQ ID NO:91 (GenBank Accession No: NC_002025.1):

(SEQ ID NO: 91) 1GTTTTAAAAC CATTTTCAAA ATATTCCAAT TCAACTCAAT TAACGCTTTT ACAGTGTAAT 61TCGTACTTTT CGTAAGTAAG TTTCTGTAAA AGCGTTTCTT GTTTTAATTT GGTCTAACAC 121GTAATTCGTA CTCTTCGTGA GTAAGTTGTG TTAGCCATAC CTATCCTTTA AATTTCTGTC 181AATTTAAAAA GAAAATCATT CCCATTTGCG TAATTCGTAC TCTTCGTGAG TAAGTTGTAA 241ATGGAGAATA CAAAAACAAA TGCCTCGAGT TCTGGAATGT CTTCTTCCTC CAGCTTTTCA 301GTGTCTTATG CTGAGGAAAT GTTACTAGCT GATGAAGTTT CAAAAATTAA CTCAATGTCG 361ATTCTGGGTC CTAATCAGCT AAAGCTCTGC ACTCAATTGG TGCTGTCTAA TGGAGCAGCG 421CCAGTAGTTT TAAGCCTTGT GTCAAAGGAA AAGAAATCGA TTTTAAATCG TATGCTTCCT 481AAGATTGGAC AGAGGATGTA CGTCCATCAC TCGGCTATTT ACCTCCTTTA TATGCCAAAC 541ATACTGAAAA GTTCTTCAGG GAGCATCACC TTGAAACTTT TTAATGAAGC TACAGGAGAG 601TTAGTGGATG TTGACACCGA CCATGATGCT ACCCAGGCAT GTATATTTGC TGGACGTTAC 661CCCCGGAGTA TTCTGGCGAA AGATGCAGCG AAAGGACACG ACTTGAAATT AGTCGTCCAC 721GCTGTTGCTT CGACCAATGC GAACTCCGCT GTCGGTGTTC TATACCCCAT TTGGGAAGAT 781GAGTTGAGCA GAAAGCAGAT CCTCGAAAGG GGTGCCGATT TCCTAAAGTT TCCAATTGCT 841GAGACCGAGC CAGTCCGCGA TCTCTTAAAT GCTGGGAAGT TGACGGACTT TGTTCTTGAT 901AGGACAAGGT TGGGTGTGGG GTCAAAGAAT GATCCCAGTC CGGTTCTTTT AGAACCAAGA 961GCTAAGATTA CCGGGAAGGC AAAGACAGTT TTTATTCCCG AAGGTCCTAG TGTTCCTAAT 1021ACCACTATAA ATGGTATGGC ACCAACGGTG CGTATAGATG CCGGTTCTCC AAAGGGTCTT 1081GGAGTTCCGA AAGGGTTTAC ATATGAAAGT TTTATTAAAG ATGAAATATT ACCCGATCAT 1141TGATCGGTAA TGGGCCGTTT TTATTTTTAA TTTTCTTTCA ATTACTTCCA TCATGAGTTC 1201TTCACAAAAG AAAGCTGGTG GGAAAGCTGG TAAACCTACT AAACGTTCTC AGAACTATGC 1261TGCCTTACGC AAAGCTCAAC TGCCGAAGCC TCCGGCGTTG AAAGTCCCGG TTGTAAAACC 1321GACGAATACT ATACTGCCAC AGACGGGCTG CGTGTGGCAA AGCCTCGGGA CCCCTCTGAG 1381TCTGAGCTCT TTTAATGGGC TCGGCGTGAG ATTCCTCTAC AGTTTTCTGA AGGATTTCGC 1441GGGACCTCGG ATCCTCGAAG AGGATCTGAT TTACAGGATG GTGTTTTCCA TAACACCGTC 1501CTATGCCGGC ACCTTTTGTC TCACTGATGA CGTGACGACT GAGGATGGTA GGGCCGTTGC 1561GCATGGTAAT CCCATGCAAG AATTTCCTCA TGGCGCGTTT CACGCTAATG AGAAGTTCGG 1621GTTTGAGTTG GTCTTCACAG CTCCTACCCA TGCGGGAATG CAAAACCAAA ATTTCAAGCA 1681TTCCTATGCC GTAGCCCTCT GTCTGGACTT CGACGCGCAG CCTGAGGGAT CTAAAAATCC 1741CTCATACCGA TTCAACGAAG TTTGGGTCGA GAGAAAGGCG TTCCCGCGAG CAGGGCCCCT 1801CCGCAGTTTG ATTACTGTGG GGCTGCTCGA CGAAGCTGAC GATCTTGATC GTCATTGATG 1861TACCCCATTA ATTTGGGATG CCAAAGTCAT TTGATGCTGA CCTCCACTGG GTGGATTAAG 1921GTCAAGGTAT GAAGTCCTAT TCGCTCCTGA TAGGATCGAC TTCATATTGC TTATATATGT 1981GCTAACGCAC ATATATAAAT GCTCATGCAA AACTGCATGA ATGCCCCTAA GGGATGC.

In embodiments, the subsequence is selected from the region of the RNA 3that encodes the coat protein, whose amino acid sequence is set forthbelow as SEQ ID NO:92 (GenBank Accession No: NP_041195.1):

(SEQ ID NO: 92) MSSSQKKAGGKAGKPTKRSQNYAALRKAQLPKPPALKVPVVKPTNTILPQTGCVWQSLGTPLSLSSFNGLGVRFLYSFLKDFAGPRILEEDLIYRMVFSITPSYAGTFCLTDDVTTEDGRAVAHGNPMQEFPHGAFHANEKFGFELVFTAPTHAGMQNQNFKHSYAVALCLDFDAQPEGSKNPSYRFNEVWVERKAFPRA GPLRSLITVGLLDEADDLDRH

Beet Curly Top Virus (BCTV)

In some embodiments, the analysis comprises detecting the presence,absence and/or amount of a Beet Curly Top Virus (BCTV) in the plantsample (e.g., a Cannabis plant sample). In some embodiments, theanalysis comprises determining one or more genotypes of a BCTV. In someembodiments, the analysis comprises determining a genetic variationsignature of a BCTV.

BCTV was first discovered in 1888 in the Western parts of the UnitedStates. The virus was not fully recognized until 1907, when peoplestarted to realize a loss in crop yield that was attributable to thevirus. In addition to the Unites States, BCTV has been known to affectother parts of the world including Mexico, South America, theMediterranean basin, and the Middle East.

BCTV has been known to affect more than 300 plant species from 44different families. It is a DNA virus containing a single-strandedcircular DNA that is encapsulated in a twinned icosahedral capsid. Thevirus DNA contains a monopartite genome that is made up of three viralsense and four complementary open reading frames (ORFS C1-C4). The ORFComplementary 1 (C1) contains the code for the replication initiatorprotein (Rep) which is responsible for initiating replication in a hostplant cell. C3 also plays an important role in the replication process.C2 is involved in causing the disease (pathogenicity), while C4 plays animportant role in developing the major symptoms that comes with thevirus, such as hyperplasia, curling of the leaves, and deformation.Symptom of infection include: vein swelling (the earliest and mostcommon symptom), leaf curling, yellowing of leaves with purple veins,necrosis and hyperplasia of the phloem, fruit deformation, prematurefruit ripening, reduced fruit quality and yield, stunting and the deathof young seedlings.

Provided herein are methods and compositions for determining thepresence, absence and/or amount of BCTV in a plant cultivar. In themethods and compositions provided herein, polynucleotide primer pairsare used to specifically hybridize to and amplify a subsequence of thenucleic acid of BCTV, or a complement thereof, where the primer pairsand/or the subsequence are non-identical to any subsequence, orcomplement thereof, of equivalent length in the nucleic acid of theplant genome. In embodiments, the subsequence of the nucleic acid ofBCTV that is amplified is a conserved sequence. In certain embodiments,the subsequence of the nucleic acid of BCTV, or a portion thereof, is ina coding region. In embodiments, the subsequence of the nucleic acid ofBCTV, or a portion thereof, is in a coding region, or in a region ofoverlap between more than one gene of the nucleic acid of the BCTV, orin a region of overlap between more than one coding region of thenucleic acid of the BCTV. For sequences of the BCTV genome and proteinsencoded therein, see, for example, GenBank Accession No: KX867057

In certain embodiments, the subsequence of the nucleic acid of the BCTVthat is amplified is a subsequence of:

(a) SEQ ID NO: 110 (Nucleic acid encodingthe SS-ds-DNA-Regulator Protein):ATGGGACCTTTCAGAGTGGATCAATTTCCAGACAATTATCCAGCCTTTCTAGCAGTATCGACCAGTTGTTTCTTAAGGTACAACAGGTGGTGTATACTAGGTATCCATCAAGAGATAGAGCCTCTGACCCTAGAAGAAGGCGAGGTCTTTCTGCAATTCCAGAAGGAAGTCAAGAAGCTACTGAGGTGTAAGGTCAACTTTCATAGGAAGTGTTCGTTGTATGAGGAAATATACAAGAAATACGTATACAATGTCCCAGAAAAGAAAGGTGAATCCTCAAAGTGCGTGGCCGAAGAAGAGGAGGACTACTACGACTTCGAGGAAATACCAATGGAGGAGACCTGTGACAAAAAACAGGAC TCCGAAGTTAAAGATGTATGA,where the SS-ds-DNA-Regulator Protein hasthe sequence set forth in SEQ ID NO: 111:MGPFRVDQFPDNYPAFLAVSTSCFLRYNRWCILGIHQEIEPLTLEEGEVFLQFQKEVKKLLRSKVNFHRKCSLYEEIYKEYVYNVPEKKGESSKCVAEEEEDYYDFEEIPMEEICDKKQDSEVKDV (SEQ ID NO: 111);(b) SEQ ID NO: 112 (Nucleic acid encoding the Movement Protein):ATGATGGTCTGTCTACCAGACTGGTTATTTTTGCTATTTATCTTCAGTATTCTACTGCAATCAGGTACCAACTTTTATGGGACCTTTCAGAGTGGATCAATTTCCAGACAATTATCCAGCCTTTCTAGCAGTATCGACCAGTTGTTTCTTAAGGTACAACAGGTGGTGTATACTAGGTATCCATCAAGAGATAGAGCCTCTGACCCTAGAAGAAGGCGAGGTCTTTCTGCAATTCCAGAAGGAAGTCAAGAAGCTACTGAGGTGTAA,where the Movement Protein has the sequence set forth in SEQ ID NO: 113:MMVCLPDWLFLLFIFSILLQSGTNFYGTFQSGSISRQLSSLSSSIDQLFLKVQQVVYTRNPSRDRASDPRRRRGLSAIPEGSEEATEV (SEQ ID NO: 113);(c) SEQ ID NO: 114 (Nucleic acid encodingthe Rolling Circle Replication Protein (RCR)):TTACAGGGGAGATTGACCTTGCGAGGACGCTTCTGTATCTTTATCAAAGAGAGGGCCGGAGAGTTTAACGAAGGTTGAATTCTGTATAGTCCAGGACCTAAGGGCTTCATTTTCTGATTTATCTAGGAAGTCCTGGTAAGAGCTGCCTTCGCCTGGATTGCATAATATAATACTAGGAATACCACCTTTAATGACACGTGGTTTTCCATACTTTAAGTTTGTCTGCCACTCTCTTTGTGCGCCTATGAGGTGTTTCCAATGCTTCATCTTTAAGTAAGCTGGGTCTACGTCATCAATGACGTTATATAAAACATCATCGTGATATGTTTTTAAACTAAAATCTAAATGGCCCGATATATAATTATGAGGTCCTAATGATCTAGCCCACATTGTTTTACCCGTTCTAGAATCACCCTCTATGATTATACTATTATATCTAAAAGGCCGCGCAGCGGCATCCACCCCGAAATAAGAGTCGGCCCATTCTTGAACAATTTCTGGAACTCGAGTGAAAGAAGATTGTGGGAATGGAGGTTGATAAATATCTGGTGGAGGAAGAAAAATGGCTTCTAAATTAGGTTTAAGGTTGTGATACTGAAAAATAAATTTTTCTGGGAGTTTCTCCCTTATTATTTGCAGTGCTTCAGCTGCATTACCTGCATTTAATGCTTCTGCTGCTGCATCATTAGCCGTCTGCTGGCCTCCTCTAGCAGATCTTCCGTCGACTTGAAATGTACCCCAGTCGACGTAATCACCGTCCTTCTCGATGTATTGTTTAACATCGGATGCAGATTTTGCTCCCTGGAAGTTGGGGTGGAAGGTGGAGCTTGAGGAAGGATGGGTGATGTCGAAGTGTCTAGGGTTTCTGAATTGTGCTTTACCTTTGAATTGGATGAGGGCGTGGAGATGCAGAGACCCATCCTGATGTTTTTCCTGGGATACTCTAATAAATAATTTATCAGATGGGCAAGGAATATTTTTCAATATTTCCAGAGCATCTTCTTTTATAACTGAACATCGTGGGTATGTGAGAAAGATATTTTTGGCTTTAATTTG AAATGAAGGTGATCGAGGCAT,where the RCR protein has the sequence set forth in SEQ ID NO: 115:MPRSPSFQIKAKNIFLTYPRCSIIKEDALEILKNIPCPSDKLFIRVSQEKHQDGSLHLHALIQFKGKAQFRNPRHFDITHPSSSSTFHPNFQGAKSASDVKQYIEKDGDYVDWGTFQVDGRSARGGQQTANDAAAEALNAGNAAEALQIIREKLPEKFIFQYHNLKPNLEAIFLPPPDIYQPPFPLSSFTRVPEIVQEWADSYFGLDPAARPFRYNSIIIEGDSRTGKTMWARCLGPHNYITGHLDFSLKTYSDNVLYNVIDDVDPNYLKMKHWKHLIGAQREWQTNLKYGKPRVIKGGIPSIILCNPGEGSSYQDFLNKSENEALRSVVTLQNSVFAKLTSPLFDNNQEAS SQDQSSL; (SEQ ID NO: 115)(d) SEQ ID NO: 116 (Nucleic acid encodingthe Pathogenesis Enhancement Protein):TTAATTGAGATTGAAGATTGACGCTCCAGTACCCAATCCAGTTGGTTCTTCAAGGCTCTCAAAAAACGGTCTCCAGTCAATGTCCTGTGTGATCCAGTTATCGTCAAATCGATCCAGCACTTGTGTAGGTTGAGCGATTTGCGGAGGTTGTGGTTGAATCTCATCTGGACTTTTAGTTGATATATCGTTCCGAATCTCTCGAACCATAGTAGTTTGAAGTAGAGTGGATTCGGAACTGATGTTGTTGGTGTTGATTTCGTCGCCTGTTCCAGGGTAATAGGTAGTTCCGTGCGAAAATCCGTGATGGCATTCATGATGAATTGTGAAGTGACACTTACAGGGGAGATTGACCTTGCGAGGACGCTTCTGTATCTTTATCAAAGAGAGGGCCGGAGAGTTTAACGAAGGTTGAATTCTGTATAGTCCAGGACCTAAGGGCT TCAT,where the Pathogenesis Enhancement Proteinhas the sequence set forth in SEQ ID NO: 117:MKPLGPGHYKIQSSPNSQVLSLITIKKRPRKINLPCKCHFTIHHECHQGFSHRGTHYSATSDEIHTRGLGTESTVPQTPGLLPYRASLSTESPDKIQPQPPQILESSQVLDRFDDHWITQDIDWRPFFESLEEPSRQGNQKTIFSLN; (SEQ ID NO: 117)(e) SEQ ID NO: 118 (Nucleic acid encodingthe Cell Cycle Regulator Protein):TTACACCTCAGTAGCTTCTTGACTTCCTTCTGGAATTGCAGAAAGACCTCGCCTTCTTCTAGGGTCAGAGGCTCTATCTCTTGATGGATACCTAGTATACACCACCTGTTGTACCTTAAGAAACAACTGGTCGATACTGCTAGAAAGGCTGGATAATTGTCTGGAAATTGATCCACTCTGAAAGGTCCCATAAAAGTTGGTACCTGATTGCAGTAGAATACTGAAGATAAATAGCAAAAATAACCAGTCTGGTAGACAGACCAT CAT,where the Cell Cycle Regulator Proteinhas the sequence set forth in SEQ ID NO: 119:MGLCISTPSSNSKVKHNSETLDTSTSLILPQAPPSTPTSREQNLHPMLNNTSRRTVITSTGVHFKSTEDLLEEASRRLMMQQQKH; (SEQ ID NO: 119)(f) SEQ ID NO: 120 (Nucleic acid encodingthe Replication Enhancer Protein):TTAATACAATTTCATTGCAATACTAGTATATTGAATTACACTACTGACGAAATTGAAACGCTTATACAATATATAATTGAAAATACGAATAATTTTATTAATTGAGATTGAAGATTGACGCTCCAGTACCCAATCCAGTTGGTTCTTCAAGGCTCTCAAAAAACGGTCTCCAGTCAATGTCCTGTGTGATCCAGTTATCGTCAAATCGATCCAGCACTTGTGTAGGTTGAGCGATTTGCGGAGGTTGTGGTTGAATCTCATCTGGACTTTTAGTTGATATATCGTTCCGAATCTCTCGAACCATAGTAGTTTGAAGTAGAGTGGATTCGGAACTGATGTTGTTGGTGTTGATTTCGTCGCCTGTTCCAGGGTAATAGGTAGTTCCGTGCGAAAATC CGTGATGGCATTCAT,where the Replication Enhancer Proteinhas the sequence set forth in SEQ ID NO: 121:MNVIRDFRTEEPITLQQATKSIPVDLVPNPLYLKLQDFFRTGPVYQLKVQIRFNHNLRKYLNLHKCWIDLTITGSHRTLTGDRFLRVLKNQVDREIKKRSSLSINIVTEILNHVLYSTFNFVNSVIQYTSIA MKLY; (SEQ ID NO: 121)orregions of overlap that span any two of SEQ ID NOS:110, 112, 114, 116,118 or 120 in the genome of BCTV. In embodiments, the subsequence of thenucleic acid of the pathogen to which the polynucleotide primer pair iscapable of hybridizing is in a region of overlap that spans:

-   -   (i) the gene encoding the SS-ds-DNA Regulator Protein (SEQ ID        NO:110) and the gene encoding Movement Protein (SEQ ID NO:112);    -   (ii) the gene encoding the Pathogenesis Enhancement Protein (SEQ        ID NO:116) and the gene encoding the Rolling Circle Replication        Protein (SEQ ID NO:114);    -   (iii) the gene encoding the Rolling Circle Replication Protein        (SEQ ID NO:114) and the gene encoding the Cell Cycle Regulator        Protein (SEQ ID NO:118); or    -   (iv) the gene encoding the Pathogenesis Enhancement Protein (SEQ        ID NO:116) and the gene encoding the Replication Enhancer        Protein (SEQ ID NO:120).

Pathogen Detection

Provided herein are methods for analyzing nucleic acid from a plantsample. Also provided herein are methods for generating nucleic acidamplification products from a plant sample. Also provided herein aremethods for preparing a nucleic acid mixture. In certain embodiments,the methods provided herein determine the presence, absence and/oramount of a pathogen in the plant sample. A method herein may comprisecontacting nucleic acid of a plant sample with a polynucleotide primerpair under amplification conditions. In some embodiments, a methodherein comprises contacting nucleic acid of a plant sample with one ormore polynucleotide primer pairs under amplification conditions. In someembodiments, a method herein comprises contacting nucleic acid of aplant sample with a plurality of polynucleotide primer pairs underamplification conditions. A plurality of primer pairs may comprise twoor more polynucleotide primer pairs, three or more polynucleotide primerpairs, four or more polynucleotide primer pairs, five or morepolynucleotide primer pairs, six or more polynucleotide primer pairs,seven or more polynucleotide primer pairs, eight or more polynucleotideprimer pairs, nine or more polynucleotide primer pairs, or ten or morepolynucleotide primer pairs. The primers described in this section may,in certain embodiments, be referred to as primary primers, a first setof primers, and/or thermomutant-resistant primers. For HpLVd, examplesof primary primers, a first set of primers, and/orthermomutant-resistant primers are provided in Table 1 (primers labeledtm-resistant). The reverse complement for each primer also iscontemplated herein.

In some embodiments, a method comprises generating one or moreamplification products. Amplification products may be generated by anysuitable amplification method described herein or known in the art(e.g., polymerase chain reaction (PCR)). Suitable amplificationconditions include any conditions that can generate an amplificationproduct, when a target nucleic acid is contacted with primers that arecapable of hybridizing to the target nucleic acid. In some embodiments,a method comprises generating a mixture (e.g., a mixture of two or moreamplification product species). A mixture of two or more amplificationproduct species may be generated when two or more primer pairs hybridizeto different regions of a target nucleic acid. Such amplificationproduct species may have different lengths and/or different nucleotidesequences, which may include overlapping and/or non-overlappingsequences.

Generally, a primer pair comprises a forward primer and a reverseprimer. Two primer pairs may comprise two different forward primerspecies (e.g., A-fwd and B-fwd) and two different reverse primer species(e.g., A-rev, B-rev); may comprise one forward primer species (e.g.,A-fwd) and two different reverse primer species (e.g., A-rev, B-rev); ormay comprise two different forward primer species (e.g., A-fwd andB-fwd) and one reverse primer species (e.g., A-rev), provided thecombination of forward and reverse primer species is capable ofgenerating two amplification product species. Further forward andreverse primer combinations are contemplated for additional primerpairs. For HpLVd, an example of forward and reverse primer pairingcombinations, with the corresponding amplification product species, isprovided in Table 2 herein.

Examples of Certain HpLVd Primer Pairs

In some embodiments, polynucleotide primer pairs hybridize tosubsequences of SEQ ID NO:1 (i.e., subsequences of the HpLVd genome).Generally, polynucleotide primer pairs hybridize to subsequences of SEQID NO:1 if the subsequences are present in the nucleic acid of a plantsample (e.g., when the plant has been infected with HpLVd). Furthermore,polynucleotide primer pairs do not hybridize to subsequences of SEQ IDNO:1 if the subsequences are not present in the nucleic acid of a plantsample (e.g., when the plant has not been infected with HpLVd). In someembodiments, when a plurality of primer pairs is used, a majority of thepolynucleotide primer pairs hybridize to subsequences of SEQ ID NO:1. Amajority of the polynucleotide primer pairs may refer to greater than50% of the primer pairs. For example, a majority of the polynucleotideprimer pairs may refer to greater than 60% of the primer pairs, greaterthan 70% of the primer pairs, greater than 80% of the primer pairs, orgreater than 90% of the primer pairs. In some embodiments, all (e.g.,100%) of the polynucleotide primer pairs hybridize to subsequences ofSEQ ID NO:1.

In some embodiments, the subsequences of SEQ ID NO:1 to which thepolynucleotide primers hybridize (also referred to as primerhybridization sites) contain no variant nucleotide position. A variantnucleotide position refers to a nucleotide (or base) at a given positionin SEQ ID NO:1 that may be mutated (e.g., during thermotherapy) and/ordiffers among various HpLVd strains (e.g., may contain a referenceallele or an alternate allele). A subsequence containing no variantposition refers to a subsequence where each base is not subject tomutation (e.g., during thermotherapy) and has no known alternativevariants (i.e., no known nucleotide substitutions, insertions, ordeletions at each position).

In some embodiments, the subsequences of SEQ ID NO:1 to which thepolynucleotide primers hybridize contain one variant nucleotideposition. A subsequence containing one variant position refers to asubsequence where one base is subject to mutation (e.g., duringthermotherapy) and/or is a known alternative variant (i.e., a knownnucleotide substitution, insertion, or deletion at the variantposition).

In some embodiments, each subsequence of SEQ ID NO:1 between thesubsequences to which the primer pairs hybridize contain one or morevariant nucleotide positions. As noted above, a variant nucleotideposition refers to a nucleotide at a given position in SEQ ID NO:1 thatmay be mutated (e.g., during thermotherapy) and/or differs among variousHpLVd strains. A subsequence of SEQ ID NO:1 between the subsequences towhich the primer pairs hybridize may be referred to as a targetsequence. A target sequence generally refers to a subsequence of SEQ IDNO:1 between two primer hybridization sites, and generally does notinclude the primer hybridization sites themselves. Thus, the variantpositions described for a target sequence do not include positions inthe primer hybridization sites. In some embodiments, a target sequencecomprises one variant nucleotide position. In some embodiments, a targetsequence comprises two or more variant nucleotide positions. In someembodiments, a target sequence comprises three or more variantnucleotide positions. In some embodiments, a target sequence comprisesfour or more variant nucleotide positions. In some embodiments, a targetsequence comprises five or more variant nucleotide positions. In someembodiments, a target sequence comprises six or more variant nucleotidepositions. In some embodiments, a target sequence comprises seven ormore variant nucleotide positions. In some embodiments, a targetsequence comprises eight or more variant nucleotide positions. In someembodiments, a target sequence comprises nine or more variant nucleotidepositions. In some embodiments, a target sequence comprises ten or morevariant nucleotide positions.

In some embodiments, each polynucleotide in each primer pair comprises asequence that is non-identical to any subsequence, or complementthereof, in a cannabis genome. In some embodiments, each polynucleotidein each primer pair comprises a sequence that is non-identical to anysubsequence, or complement thereof, in a Cannabis sativa genome,Cannabis indica genome, or Cannabis ruderalis genome. Examples ofcannabis genomes include CS10, Arcata Trainwreck, Grape Stomper, Citrix,Black 84, Headcheese, Red Eye OG, Tahoe OG, Master Kush, Chem 91,Domnesia, Sour Tsunami, Sour Tsunami_x_CK, Tibor_1_2016, 80 E-1, 80 E-2,80 E-3, Harlox, Saint Jack, Herijuana, Mothers Milk_5, Black Beauty,Sour Diesel, JL_1, JL_2, JL_3, JL_4, JL_5, JL_6, JL_father,BBCC_x_JL_father, JL_mother, JL_mother_p, IdaliaFT_1, Fedora17_6_1,Carmal_1_2016, CS_1_2016, EICam_1_2016, C3/USO-1, Carmagnola_3, andMerino_S_1. In some embodiments, each polynucleotide in each primer paircomprises a sequence that is non-identical to any subsequence, orcomplement thereof, in a CS10 Cannabis genome (GENBANK assemblyaccession: GCA_900626175.1; REFSEQ assembly accession: GCF_900626175.1).

A sequence that is non-identical to any subsequence, or complementthereof, in a Cannabis genome generally refers to a sequence comprisingone or more mismatched nucleotides when compared to any subsequence, orcomplement thereof, in a Cannabis genome (e.g., CS10 Cannabis genome).In some embodiments, each polynucleotide in each primer pair comprises asequence comprising at least two mismatches when compared to anysubsequence, or complement thereof, in a cannabis genome (e.g., CS10Cannabis genome). In some embodiments, each polynucleotide in eachprimer pair comprises a sequence comprising at least three mismatcheswhen compared to any subsequence, or complement thereof, in a cannabisgenome (e.g., CS10 Cannabis genome). In some embodiments, eachpolynucleotide in each primer pair comprises a sequence comprising atleast four mismatches when compared to any subsequence, or complementthereof, in a cannabis genome (e.g., CS10 Cannabis genome). In someembodiments, each polynucleotide in each primer pair comprises asequence comprising at least five mismatches when compared to anysubsequence, or complement thereof, in a cannabis genome (e.g., CS10Cannabis genome). In some embodiments, each polynucleotide in eachprimer pair comprises a sequence comprising at least six mismatches whencompared to any subsequence, or complement thereof, in a Cannabis genome(e.g., CS10 Cannabis genome). In some embodiments, each polynucleotidein each primer pair comprises a sequence comprising at least sevenmismatches when compared to any subsequence, or complement thereof, in acannabis genome (e.g., CS10 Cannabis genome). In some embodiments, eachpolynucleotide in each primer pair comprises a sequence comprising atleast eight mismatches when compared to any subsequence, or complementthereof, in a cannabis genome (e.g., CS10 Cannabis genome). In someembodiments, each polynucleotide in each primer pair comprises asequence comprising at least nine mismatches when compared to anysubsequence, or complement thereof, in a Cannabis genome (e.g., CS10Cannabis genome). In some embodiments, each polynucleotide in eachprimer pair comprises a sequence comprising at least ten mismatches whencompared to any subsequence, or complement thereof, in a Cannabis genome(e.g., CS10 Cannabis genome).

The primers provided herein generally share a high degree of sequenceidentity to a subsequence, or complement thereof, of SEQ ID NO:1. Insome embodiments, each polynucleotide in each primer pair comprises asequence that is at least about 90% identical to a subsequence, orcomplement thereof, of SEQ ID NO:1. In some embodiments, eachpolynucleotide in each primer pair comprises a sequence that is at leastabout 95% identical to a subsequence, or complement thereof, of SEQ IDNO:1. In some embodiments, each polynucleotide in each primer paircomprises a sequence that is 100% identical to a subsequence, orcomplement thereof, of SEQ ID NO:1.

The primers provided herein generally hybridize to regions of the HpLVdgenome that are free of thermomutant sites (i.e., nucleotide positionssusceptible to mutation under heat treatment conditions). Such primersmay be referred to as thermomutant-resistant primers. Example regions ofthe HpLVd genome that are free of thermomutant sites include thesubsequence between nucleotide position 60 and nucleotide position 102of SEQ ID NO:1, the subsequence between nucleotide position 89 andnucleotide position 119 of SEQ ID NO:1, and subsequence betweennucleotide position 178 and nucleotide position 198 of SEQ ID NO:1. Insome embodiments, each forward primer hybridizes to a subsequencebetween nucleotide position 60 and nucleotide position 102 of SEQ IDNO:1. In some embodiments, each reverse primer hybridizes to asubsequence between nucleotide position 89 and nucleotide position 119of SEQ ID NO:1, or hybridizes to a subsequence between nucleotideposition 178 and nucleotide position 198 of SEQ ID NO:1.

In some embodiments, the subsequences of SEQ ID NO:1 to which thepolynucleotide primers hybridize (i.e., primer hybridization sites)contain no thermomutant positions (thermomutant sites). Thermomutantpositions may be chosen from one or more of nucleotide position 7 of SEQID NO:1, nucleotide position 10 of SEQ ID NO:1, nucleotide position 12of SEQ ID NO:1, nucleotide position 26 of SEQ ID NO:1, nucleotideposition 27 of SEQ ID NO:1, nucleotide position 28 of SEQ ID NO:1,nucleotide position 29 of SEQ ID NO:1, nucleotide position 30 of SEQ IDNO:1, nucleotide position 33 of SEQ ID NO:1, nucleotide position 35 ofSEQ ID NO:1, nucleotide position 43 of SEQ ID NO:1, nucleotide position59 of SEQ ID NO:1, nucleotide position 121 of SEQ ID NO:1, nucleotideposition 128 of SEQ ID NO:1, nucleotide position 134 of SEQ ID NO:1,nucleotide position 150 of SEQ ID NO:1, nucleotide position 157 of SEQID NO:1, nucleotide position 162 of SEQ ID NO:1, nucleotide position 168of SEQ ID NO:1, nucleotide position 169 of SEQ ID NO:1, nucleotideposition 177 of SEQ ID NO:1, nucleotide position 200 of SEQ ID NO:1,nucleotide position 225 of SEQ ID NO:1, nucleotide position 229 of SEQID NO:1, nucleotide position 247 of SEQ ID NO:1, nucleotide position 248of SEQ ID NO:1, and nucleotide position 253 of SEQ ID NO:1.

Forward primers provided herein (i.e., thermomutant-resistant forwardprimers) generally share a high degree of sequence identity to asubsequence, or complement thereof, of SEQ ID NO:1. In some embodiments,one or more forward primers (i.e., one or more thermomutant-resistantforward primers) independently are chosen from a polynucleotidecomprising a sequence that is at least about 90% identical toGGGGAAACCTACTCGAGCG (SEQ ID NO:4), GGAAACCTACTCGAGCGAGGCG (SEQ ID NO:6),CGAGGCGGAGATCGAGCGC (SEQ ID NO:9), GAGATCGAGCGCCAGTTCG (SEQ ID NO:11),and AGATCGAGCGCCAGTTCG (SEQ ID NO:13). In some embodiments, one or moreforward primers (i.e., one or more thermomutant-resistant forwardprimers) independently are chosen from a polynucleotide comprising asequence that is at least about 95% identical to GGGGAAACCTACTCGAGCG(SEQ ID NO:4), GGAAACCTACTCGAGCGAGGCG (SEQ ID NO:6), CGAGGCGGAGATCGAGCGC(SEQ ID NO:9), GAGATCGAGCGCCAGTTCG (SEQ ID NO:11), andAGATCGAGCGCCAGTTCG (SEQ ID NO:13). In some embodiments, one or moreforward primers (i.e., one or more thermomutant-resistant forwardprimers) independently are chosen from a polynucleotide comprising asequence that is 100% identical to GGGGAAACCTACTCGAGCG (SEQ ID NO:4),GGAAACCTACTCGAGCGAGGCG (SEQ ID NO:6), CGAGGCGGAGATCGAGCGC (SEQ ID NO:9),GAGATCGAGCGCCAGTTCG (SEQ ID NO:11), and AGATCGAGCGCCAGTTCG (SEQ IDNO:13).

Reverse primers provided herein (i.e., thermomutant-resistant reverseprimers) generally share a high degree of sequence identity to asubsequence, or complement thereof, of SEQ ID NO:1. In some embodiments,one or more reverse primers (i.e., one or more thermomutant-resistantreverse primers) independently are chosen from a polynucleotidecomprising a sequence that is at least about 90% identical toCGCACGAACTGGCGCTCG (SEQ ID NO:3), CTTCAGGTCGCCGCGCACG (SEQ ID NO:5),CGGGTAGTTTCCAACTCCG (SEQ ID NO:8), CCGGGTAGTTTCCAACTCCG (SEQ ID NO:10),and ACCGGGTAGTTTCCAACTCCG (SEQ ID NO:12). In some embodiments, one ormore reverse primers (i.e., one or more thermomutant-resistant reverseprimers) independently are chosen from a polynucleotide comprising asequence that is at least about 95% identical to CGCACGAACTGGCGCTCG (SEQID NO:3), CTTCAGGTCGCCGCGCACG (SEQ ID NO:5), CGGGTAGTTTCCAACTCCG (SEQ IDNO:8), CCGGGTAGTTTCCAACTCCG (SEQ ID NO:10), and ACCGGGTAGTTTCCAACTCCG(SEQ ID NO:12). In some embodiments, one or more reverse primers (i.e.,one or more thermomutant-resistant reverse primers) independently arechosen from a polynucleotide comprising a sequence that is 100%identical to CGCACGAACTGGCGCTCG (SEQ ID NO:3), CTTCAGGTCGCCGCGCACG (SEQID NO:5), CGGGTAGTTTCCAACTCCG (SEQ ID NO:8), CCGGGTAGTTTCCAACTCCG (SEQID NO:10), and ACCGGGTAGTTTCCAACTCCG (SEQ ID NO:12).

A plurality of polynucleotide primer pairs generally comprises aplurality of forward primers and a plurality of reverse primers. In someembodiments, a plurality of forward primers comprisesGGGGAAACCTACTCGAGCG (SEQ ID NO:4), GGAAACCTACTCGAGCGAGGCG (SEQ ID NO:6),CGAGGCGGAGATCGAGCGC (SEQ ID NO:9), GAGATCGAGCGCCAGTTCG (SEQ ID NO:11),and AGATCGAGCGCCAGTTCG (SEQ ID NO:13); and a plurality of reverseprimers comprises CGCACGAACTGGCGCTCG (SEQ ID NO:3), CTTCAGGTCGCCGCGCACG(SEQ ID NO:5), CGGGTAGTTTCCAACTCCG (SEQ ID NO:8), CCGGGTAGTTTCCAACTCCG(SEQ ID NO:10), ACCGGGTAGTTTCCAACTCCG (SEQ ID NO:12), andAGAGTTGTATTCACCGGGTAGTTTCC (SEQ ID NO:14). In some embodiments, aplurality of forward primers consists of GGGGAAACCTACTCGAGCG (SEQ IDNO:4), GGAAACCTACTCGAGCGAGGCG (SEQ ID NO:6), CGAGGCGGAGATCGAGCGC (SEQ IDNO:9), GAGATCGAGCGCCAGTTCG (SEQ ID NO:11), and AGATCGAGCGCCAGTTCG (SEQID NO:13); and a plurality of reverse primers consists ofCGCACGAACTGGCGCTCG (SEQ ID NO:3), CTTCAGGTCGCCGCGCACG (SEQ ID NO:5),CGGGTAGTTTCCAACTCCG (SEQ ID NO:8), CCGGGTAGTTTCCAACTCCG (SEQ ID NO:10),and ACCGGGTAGTTTCCAACTCCG (SEQ ID NO:12).

In certain embodiments, an additional example of a thermomutant-specificpolynucleotide primer pair is as follows:

(Forward Primer) HpLVd_1-Fwd: (SEQ ID NO: 77) GTGACTTACCTGTATGGTGGCAA(Reverse Primer) HpLVd_1-Rev: (SEQ ID NO: 78) CTCGCTCGAGTAGGTTTCCCC

In embodiments, the amplicon generated by amplifying a subsequence ofthe HpLVd genome is quantitated using the polynucleotide probe havingthe following sequence:

HpLVd_1-Probe: (SEQ ID NO: 79) GGGCTCGAAGAGGGATCOCC

The specifications for the above polynucleotide primer pair (SEQ IDNOS:77 and 78) and the above polynucleotide probe (SEQ ID NO:79) are setforth in Table 16 below:

TABLE 16 Self Self 3′ Se- Temp- comple- comple- quence late GC mentar-mentar- (5′->3′) strand Length Start Stop Tm % ity ity HpLVd_ GTGACTTPlus 23 17 39 60.56 47.83 4.00 2.00 1- ACCTGT Fwd ATGGTG GCAA (SEQ IDNO: 77) HpLVd_ CTCGCT Minus 21 80 60 62.22 61.90 6.00 0.00 1- CGAGTA RevGGTTTC CCC (SEQ ID NO: 78) HpLVd_ GGGCTC Plus 20 40 59 57.93 70.00 1-GAAGAG probe GGATCC CC (SEQ ID NO: 79) Product 64 length

Examples of Certain AMV Primer Pairs

In some embodiments, polynucleotide primer pairs hybridize tosubsequences of the AMV genome. In embodiments, polynucleotide primerpairs hybridize to subsequences of SEQ ID NO:91. Generally,polynucleotide primer pairs hybridize to subsequences of SEQ ID NO:91 ifthe subsequences are present in the nucleic acid of a plant sample(e.g., when the plant has been infected with AMV). Furthermore,generally, polynucleotide primer pairs substantially do not hybridize tosubsequences of SEQ ID NO:91 if the subsequences are not present in thenucleic acid of a plant sample (e.g., when the plant has not beeninfected with AMV). In some embodiments, when a plurality of primerpairs is used, a majority of the polynucleotide primer pairs hybridizeto subsequences of SEQ ID NO:91. A majority of the polynucleotide primerpairs may refer to greater than 50% of the primer pairs. For example, amajority of the polynucleotide primer pairs may refer to greater than60% of the primer pairs, greater than 70% of the primer pairs, greaterthan 80% of the primer pairs, or greater than 90% of the primer pairs.In some embodiments, all (e.g., 100%) of the polynucleotide primer pairshybridize to subsequences of SEQ ID NO:91.

In some embodiments, each polynucleotide in each primer pair comprises asequence that is non-identical to any subsequence, or complementthereof, in a cannabis genome. In some embodiments, each polynucleotidein each primer pair comprises a sequence that is non-identical to anysubsequence, or complement thereof, in a Cannabis sativa genome,Cannabis indica genome, or Cannabis ruderalis genome. Examples ofcannabis genomes include CS10, Arcata Trainwreck, Grape Stomper, Citrix,Black 84, Headcheese, Red Eye OG, Tahoe OG, Master Kush, Chem 91,Domnesia, Sour Tsunami, Sour Tsunami_x_CK, Tibor_1_2016, 80 E-1, 80 E-2,80 E-3, Harlox, Saint Jack, Herijuana, Mothers Milk_5, Black Beauty,Sour Diesel, JL_1, JL_2, JL_3, JL_4, JL_5, JL_6, JL_father,BBCC_x_JL_father, JL_mother, JL_mother_p, IdaliaFT_1, Fedora17_6_1,Carmal_1_2016, CS_1_2016, EICam_1_2016, C3/USO-1, Carmagnola_3, andMerino_S_1. In some embodiments, each polynucleotide in each primer paircomprises a sequence that is non-identical to any subsequence, orcomplement thereof, in a CS10 Cannabis genome (GENBANK assemblyaccession: GCA_900626175.1; REFSEQ assembly accession: GCF_900626175.1).

The primers provided herein generally share a high degree of sequenceidentity to a subsequence, or complement thereof, of SEQ ID NO:91. Insome embodiments, each polynucleotide in each primer pair comprises asequence that is at least about 90% identical, or between about 90% toabout 100% identical, to a subsequence, or complement thereof, of SEQ IDNO:91. In some embodiments, each polynucleotide in each primer paircomprises a sequence that is at least about 95%, 96%, 97%, 98% or 99%identical to a subsequence, or complement thereof, of SEQ ID NO:91. Insome embodiments, each polynucleotide in each primer pair comprises asequence that is 100% identical to a subsequence, or complement thereof,of SEQ ID NO:91.

In certain embodiments, the primer pairs that hybridize to subsequencesof SEQ ID NO:91 are shown in Table 12 below:

TABLE 12 Sequence (5′->3′) Length Start Stop A-fwd TTGGTCTT 21 1628 1648CACAGCTC CTACC (SEQ ID NO: 80) A-rev AAGTCCAG 21 1710 1690 ACAGAGGGCTACG (SEQ ID NO: 81) B-fwd CTCCTACC 22 1641 1659 CATGCGGG AAT (SEQ IDNO: 82) B-rev TCTCTCGA 19 1774 1753 CCCAAACT TCGTTG (SEQ ID NO: 83)C-rev TCGTTGAA 20 1758 1738 TCGGTATG AGGGA (SEQ ID NO: 84) D-fwdTAGGACAA 20 900 919 GGTTGGGT GTGG (SEQ ID NO: 85) D-rev GTCTTTGC 22 986965 CTTCCCGG TAATCT (SEQ ID NO: 86)

Examples of lengths of amplicons that can be generated usingcombinations of forward and reverse primers from among those set forthin Table 12 above are shown in Table 13, below:

TABLE 13 Arev Brev Crev Afwd 82 146 130 Bfwd 69 133 117

In certain embodiments, the amplicons that are generated are quantified.In embodiments, the amplicons are quantified by RT-qPCR or by qPCR. Inembodiments, the polynucleotide probes for quantifying the ampliconsgenerated by hybridizing polynucleotide primer pairs to subsequences ofSEQ ID NO:91 are as shown below in Table 14:

TABLE 14 Sequence (SEQ ID NO) Start Stop Probe A TGCGGG 1651 1676 AATGCAAAACCA AAATTT CA (87) Probe A- TGCGGG 1651 1676 degen AATGCA AAAYCAAAATTT CA (88) Probe B GAYGCGC 1712 1731 AGCCTGA GGGAT C (89) Probe DGGTCAAA 920 944 GAATGAT CCCAGTC CGGT (90)

Examples of Certain BCTV Primer Pairs

In some embodiments, polynucleotide primer pairs hybridize tosubsequences of the BCTV genome. In embodiments, polynucleotide primerpairs hybridize to subsequences of SEQ ID NOS:110, 112, 114, 116, 118 or120, or a portion of SEQ ID NOS:110, 112, 114, 116, 118 or 120, or acomplement of SEQ ID NOS:110, 112, 114, 116, 118 or 120, or a portion ofthe complement of SEQ ID NOS:110, 112, 114, 116, 118 or 120, or toregions of overlap that span more than one of SEQ ID NOS:110, 112, 114,116, 118 or 120 in the genome of the pathogen. Generally, polynucleotideprimer pairs hybridize to any of the subsequences of the BCTV genome ifthe subsequences are present in the nucleic acid of a plant sample(e.g., when the plant has been infected with BCTV). Furthermore,generally, polynucleotide primer pairs substantially do not hybridize tosubsequences of the BCTV genome if the subsequences are not present inthe nucleic acid of a plant sample (e.g., when the plant has not beeninfected with BCTV). In some embodiments, when a plurality of primerpairs is used, a majority of the polynucleotide primer pairs hybridizeto subsequences of the BCTV genome. A majority of the polynucleotideprimer pairs may refer to greater than 50% of the primer pairs. Forexample, a majority of the polynucleotide primer pairs may refer togreater than 60% of the primer pairs, greater than 70% of the primerpairs, greater than 80% of the primer pairs, or greater than 90% of theprimer pairs. In some embodiments, all (e.g., 100%) of thepolynucleotide primer pairs hybridize to subsequences of SEQ ID NO:91.

In some embodiments, each polynucleotide in each primer pair comprises asequence that is non-identical to any subsequence, or complementthereof, in a Cannabis genome. In some embodiments, each polynucleotidein each primer pair comprises a sequence that is non-identical to anysubsequence, or complement thereof, in a Cannabis sativa genome,Cannabis indica genome, or Cannabis ruderalis genome. Examples ofcannabis genomes include CS10, Arcata Trainwreck, Grape Stomper, Citrix,Black 84, Headcheese, Red Eye OG, Tahoe OG, Master Kush, Chem 91,Domnesia, Sour Tsunami, Sour Tsunami_x_CK, Tibor_1_2016, 80 E-1, 80 E-2,80 E-3, Harlox, Saint Jack, Herijuana, Mothers Milk_5, Black Beauty,Sour Diesel, JL_1, JL_2, JL_3, JL_4, JL_5, JL_6, JL_father,BBCC_x_JL_father, JL_mother, JL_mother_p, IdaliaFT_1, Fedora17_6_1,Carmal_1_2016, CS_1_2016, EICam_1_2016, C3/USO-1, Carmagnola_3, andMerino_S_1. In some embodiments, each polynucleotide in each primer paircomprises a sequence that is non-identical to any subsequence, orcomplement thereof, in a CS10 Cannabis genome (GENBANK assemblyaccession: GCA_900626175.1; REFSEQ assembly accession: GCF_900626175.1).

The primers provided herein generally share a high degree of sequenceidentity to a subsequence, or complement thereof, of SEQ ID NO:110, 112,114, 116, 118 or 120, or a portion of SEQ ID NO:110, 112, 114, 116, 118or 120, or a complement of SEQ ID NO:110, 112, 114, 116, 118 or 120, ora portion of the complement of SEQ ID NOS:110, 112, 114, 116, 118 or120, or to regions of overlap that span more than one of SEQ ID NOS:110,112, 114, 116, 118 or 120 in the genome of the BCTV pathogen. In someembodiments, each polynucleotide in each primer pair comprises asequence that is at least about 90% identical, or between about 90% toabout 100% identical, to a subsequence, or complement thereof, of SEQ IDNO:110, 112, 114, 116, 118 or 120, or a portion of SEQ ID NO:110, 112,114, 116, 118 or 120, or a complement of SEQ ID NO:110, 112, 114, 116,118 or 120, or a portion of the complement of SEQ ID NOS:110, 112, 114,116, 118 or 120, or to regions of overlap that span more than one of SEQID NOS:110, 112, 114, 116, 118 or 120 in the genome of the BCTVpathogen. In some embodiments, each polynucleotide in each primer paircomprises a sequence that is at least about 95%, 96%, 97%, 98% or 99%identical to a subsequence, or complement thereof, of of SEQ ID NO:110,112, 114, 116, 118 or 120, or a portion of SEQ ID NO:110, 112, 114, 116,118 or 120, or a complement of SEQ ID NO:110, 112, 114, 116, 118 or 120,or a portion of the complement of SEQ ID NOS:110, 112, 114, 116, 118 or120, or to regions of overlap that span more than one of SEQ ID NOS:110,112, 114, 116, 118 or 120 in the genome of the BCTV pathogen. In someembodiments, each polynucleotide in each primer pair comprises asequence that is 100% identical to a subsequence, or complement thereof,of of SEQ ID NO:110, 112, 114, 116, 118 or 120, or a portion of SEQ IDNO:110, 112, 114, 116, 118 or 120, or a complement of SEQ ID NO:110,112, 114, 116, 118 or 120, or a portion of the complement of SEQ IDNOS:110, 112, 114, 116, 118 or 120, or to regions of overlap that spanmore than one of SEQ ID NOS:110, 112, 114, 116, 118 or 120 in the genomeof the BCTV pathogen.

In embodiments, the subsequence of the nucleic acid of BCTV to which thepolynucleotide primer pair hybridizes is in a region of overlap thatspans:

-   -   (i) the gene encoding the SS-ds-DNA Regulator Protein (SEQ ID        NO:110) and the gene encoding Movement Protein (SEQ ID NO:112);    -   (ii) the gene encoding the Pathogenesis Enhancement Protein (SEQ        ID NO:116) and the gene encoding the Rolling Circle Replication        Protein (SEQ ID NO:114);    -   (iii) the gene encoding the Rolling Circle Replication Protein        (SEQ ID NO:114) and the gene encoding the Cell Cycle Regulator        Protein (SEQ ID NO:118); or    -   (iv) the gene encoding the Pathogenesis Enhancement Protein (SEQ        ID NO:116) and the gene encoding the Replication Enhancer        Protein (SEQ ID NO:120).

In certain embodiments, the polynucleotide primer pairs, and thepolynucleotide probe sequences for quantitating the resulting amplicons,are shown in Table 15 below:

**TABLE 15 Sequence (5′->3′) (SEQ ID NO) Length Start Stop Fwd_DRP_MPGACCTTTCA 25 334 358 GAGTGGATC AATTTCC (93) Rev_DRP_MP GAAAGACCT 23 480458 CGCCTTCTT CTAGG (94) Rev-2_DRP_ GMAGAAAGA MP_Degen CCTCGCCTTCT (105) Probe_DRP_MP CCAGCCTTT 25 369 393 CTAGCAGTR TCGACC A (95)Probe-2_DRP_ CCATCAAGA MP_Dege n GATAGAGSC TCTGACC C (106) Fwd_PE_GCGAGGACG 21 1781 1801 RCRI CTTCTGTA TCTT (96) Degen_Rev_ AAGCMCTTA 241857 1844 PE_RCRI RGTCCTGGA CTATA C (97) Degen_ GGGCYGGAG 23 1813 1835Probe_PE_ AGTTTAACG RCRI AAGG Y (98) Fwd_RCRI_ GCTGCATC 20 2437 2456 CCRATTAGCCG TCTG (99) Degen_ CCTTCCAC 20 2581 2562 Rev_RCRI_ CSCAACTT CCRCCAR (100) Probe_RCRI_ ACCCCAGT 23 2496 2518 CCR CGACGTAA TCACCG T (101)Fwd_PE_RE AGCGATTT 19 1559 1577 GCGGAGGT TGT (102) Rev_PE_RE AACAGGCG 201694 1675 ACGAAATC AACA (103) Probe_PE_RE AGTGGATT 26 1649 1874 CGGAACTGATGTTGTT GG (104) **DRP_MP primers and probe: targeting region ofoverlap between gene encoding the SS-ds-DNA Regulator Protein (SEQ IDNO: 110) and the gene encoding Movement Protein (SEQ ID NO: 112).PE_RCRI primers and probe: targeting region of overlap between geneencoding the Pathogenesis Enhancement Protein (SEQ ID NO: 116) and thegene encoding the Rolling Circle Replication Protein (SEQ ID NO: 114).

Certain Primers that Hybridize to Subsequences of the Plant Genome

In embodiments of the methods provided herein, a positive controlamplicon is generated using a polynucleotide primer pair that is capableof specifically hybridizing to and amplifying a subsequence of thenucleic acid of the plant genome, or to a complement thereof, whereinthe subsequence of the nucleic acid of the plant genome, or thecomplement thereof, is non-identical to any subsequence of the nucleicacid of the pathogen, or to any complement thereof; and determining thepresence, absence and/or amount of at least one amplicon that is anamplification product of the polynucleotide primer pair that is capableof specifically hybridizing to and amplifying a subsequence of thenucleic acid of the plant genome, thereby determining whether theamplification conditions are effective for generating amplicons. Inembodiments, the subsequence of the nucleic acid of the plant genomecomprises all or part of a gene selected from among 26S rRNA,beta-tubulin, ATP Synthase, an rRNA subunit, glyceraldehyde-3-phosphatedehydrogenase, Ubiquitin-conjugating enzyme E2, eukaryotic transcriptionfactors, eukaryotic initiation factor 1 and beta-actin. In embodiments,the plant genome is a Cannabis genome.

In embodiments, the polynucleotide primer pair hybridizes to asubsequence of 26S rRNA. An example of a polynucleotide primer pair thathybridizes to a subsequence of 26S rRNA is the following:

Forward Primer 26_S_Fwd: (SEQ ID NO: 107) AGAAGGGTTCGAGTGAGAGCReverse Primer 26_S_Rev: (SEQ ID NO: 108) GAGGGAAACTTCGGAGGGAA

In certain embodiments, the amplicon generated by hybridizing to andamplifying a subsequence of 26S rRNA are quantified using apolynucleotide probe (e.g., by RT-qPCR or qPCR). An example of a 26SrRNA polynucleotide probe sequence is as follows:

26S probe: (SEQ ID NO: 109) ATCGCTGCGGGCCTCCACCA.

Methods for Analyzing Nucleic Acids

Provided herein are methods for analyzing nucleic acids. In embodiments,the methods are for analyzing nucleic acids to determine the presence,absence and/or amount of a plant pathogen in a plant. The nucleic acidscan be analyzed using a variety of methods that include, but are notlimited to, RT-qPCR, qPCR, RT-PCR, and PCR ran on cDNA. The genotype ofthe plant pathogen can be determined using, e.g., amplified nucleicacids (low level or high level amplification) and/or high resolutionmelting analysis (HRM). A high-resolution melting (HRM) endpoint assayusing the polynucleotide primer pairs that specifically hybridize to andamplify a subsequence of the nucleic acid from a pathogen, as providedherein, can permit genetic classification of the variant of the pathogen(e.g., HpLVd, AMV, BCTV or any combination thereof) that infects a plantcultivar. These primers can be used as molecular markers to identify,e.g., symptomatic vs asymptomatic pathogenic variants, as well asidentify, e.g., pathogenic variants that spread more easily orpathogenic variants to which the plants have acquired resistance. Themethods provided herein can be used analyze a single plant pathogenusing a single polynucleotide primer pair and a single polynucleotideprobe, or can be performed as a multiplexed method for analyzing one ormore of: (a) a single polynucleotide primer pair and more than onepolynucleotide probe sequence for analyzing a pathogen; differences inthe Cq values that might be obtained using the different probes canprovide information regarding possible mutations (genotypic variants) inthe pathogen; (b) more than one polynucleotide primer pair to analyzemore than one non-overlapping subsequence (including, in embodiments, apolynucleotide probe sequence for each non-overlapping subsquence) of apathogen; differences in the Cq values that might be obtained for thepolynucleotide probes can provide information regarding possiblemutations (genotypic variants) in the pathogen; (c) more than onepolynucleotide primer pair to simultaneously analyze more than onepathogen that may have infected the plant, e.g., one or more of amongHops Latent Viroid (HpLVd), Alfalfa Mosaic Virus (AMV), Beet Curly TopVirus (BCTV), Hemp Streak Virus (HSV), Hemp Mosaic Virus (HMV), Tomatospotted wilt virus (TSWV), Sunn-Hemp Mosaic Virus (SHMV), Arabis MosaicVirus (ArMV), Cucumber Mosaic Virus (CMV), Lettuce Chlorosis Virus(LCV), Tobacco Ringspot Virus (TRSV), Tomato Ringspot Virus (TomRSV),and Tobacco Streak Virus (TSV), Cannabis Cryptic Virus (CCV), PotatoSpindle Tubular Viroid (PSTV), Coconut cadang cadang viroid (CCCV),Apple scar skin viroid (ASSV), Avocado sunblotch viroid (ASBV), Tobaccostreak virus (TSV), Tomato mosaic virus (ToMV), Euonymous Ringspot Virus(ERSV), Elm Mosaic Virus (EMV), and Hops Stunting Virus (HpSV).

In embodiments, the presence or absence of a wild-type or genotypicvariant pathogen in a plant, as identified by the methods providedherein, can be correlated to susceptibility of the plant to infection bythe wild-type pathogen and/or genotypic variants thereof, e.g., whetherthe plant is infected and symptomatic, infected but asymptomatic, oraltogether resistant to infection. In aspects, if the plant isidentified as resistant to infection or by the pathogen and/or agenotypic variant thereof, or asymptomatic, the plant is identified asdesirable for breeding, or as desirable for cultivating as a crop. Inaspects, the methods provided herein can be used as a way to produce,such as by self-breeding, inbreeding, and outcrossing, offspring thatare resistant to infection by a pathogen or an identified geneticvariant thereof. For example, when two plants that have latentinfections of HpLVd (infected but asymptomatic) are bred, about 8% ofthe progeny are resistant to HpLVd infection. Selective breeding andselection by identifying pathogen-resistant or asymptomatic plantsaccording to the methods provided herein can, in aspects, be used to“clean” a field containing infected plants by gradually replacing suchplants with resistant or asymptomatic progeny plants. In aspects, theplant is of the Rosidae family. In certain aspects, the plant is aCannabis plant.

In some embodiments, methods herein comprise analyzing nucleic acid froma plant sample. In some embodiments, methods herein comprise analyzingnucleic acid from a Cannabis plant sample. In some embodiments, methodsherein comprise analyzing nucleic acid from a pathogen. In someembodiments, methods herein comprise analyzing nucleic acid from apathogen that has infected a plant. In some embodiments, methods hereincomprise analyzing nucleic acid from a pathogen that is a virus selectedfrom among Hops Latent Viroid (HpLVd), Alfalfa Mosaic Virus (AMV), BeetCurly Top Virus (BCTV), Hemp Streak Virus (HSV), Hemp Mosaic Virus(HMV), Tomato spotted wilt virus (TSWV), Sunn-Hemp Mosaic Virus (SHMV),Arabis Mosaic Virus (ArMV), Cucumber Mosaic Virus (CMV), LettuceChlorosis Virus (LCV), Tobacco Ringspot Virus (TRSV), Tomato RingspotVirus (TomRSV), and Tobacco Streak Virus (TSV), Cannabis Cryptic Virus(CCV), Potato Spindle Tubular Viroid (PSTV), Coconut cadang cadangviroid (CCCV), Apple scar skin viroid (ASSV), Avocado sunblotch viroid(ASBV), Tobacco streak virus (TSV), Tomato mosaic virus (ToMV),Euonymous Ringspot Virus (ERSV), Elm Mosaic Virus (EMV), and HopsStunting Virus (HpSV). In some embodiments, methods herein compriseanalyzing nucleic acid from a Hops Latent Viroid that has infected aCannabis plant. In some embodiments, methods herein comprise analyzingnucleic acid from one or more viruses selected from among a Hops LatentViroid (HpLVd), Alfalfa Mosaic Virus (AMV) and Beet Curly Top Virus(BCTV) that has infected a plant. In embodiments, the plant is aCannabis plant.

In some embodiments, the plant (e.g., a Cannabis plant) has beensubjected to thermotherapy (has been heat treated). In embodiments, thepathogen is hops latent viroid (HpLVd). In some embodiments, the plant(e.g., a Cannabis plant) has not been subjected to thermotherapy (hasnot been heat treated). Thermotherapy (or heat treatment) generallyrefers to a process of maintaining living plants in a chamber or roomwhere light and temperature can be manipulated throughout a 24 hour timeperiod, typically providing long days of light and temperatures near100° F. for at least 16 hours and typically a lower temperature (such as25° C. to 40° C.) during the dark period. Often the conditions areadjusted as appropriate to maintain the genetics of the plant beingtreated with the goal of causing virus escape when explants are removedfrom the plants after the heating period.

In some embodiments, analyzing comprises detecting the presence orabsence and/or amount of one or more pathogens in a plant. A plant maybe a Cannabis plant. A pathogen may be a Hops Latent Viroid (HpLVd),Alfalfa Mosaic Virus (AMV), Beet Curly Top Virus (BCTV), Hemp StreakVirus (HSV), Hemp Mosaic Virus (HMV), Tomato spotted wilt virus (TSWV),Sunn-Hemp Mosaic Virus (SHMV), Arabis Mosaic Virus (ArMV), CucumberMosaic Virus (CMV), Lettuce Chlorosis Virus (LCV), Tobacco RingspotVirus (TRSV), Tomato Ringspot Virus (TomRSV), and Tobacco Streak Virus(TSV), Cannabis Cryptic Virus (CCV), Potato Spindle Tubular Viroid(PSTV), Coconut cadang cadang viroid (CCCV), Apple scar skin viroid(ASSV), Avocado sunblotch viroid (ASBV), Tobacco streak virus (TSV),Tomato mosaic virus (ToMV), Euonymous Ringspot Virus (ERSV), Elm MosaicVirus (EMV), and Hops Stunting Virus (HpSV), or any combination thereof.

HpLVd

A plant may be a Cannabis plant. A pathogen may be a Hops Latent Viroid(HpLVd). Accordingly, in some embodiments, analyzing comprises detectingthe presence or absence of a hops latent viroid (HpLVd) in a Cannabisplant. Presence of a hops latent viroid (HpLVd) in a Cannabis plant maybe determined according to amplification products generated using one ormore primer pairs that specifically amplify subsequences of a hopslatent viroid (HpLVd) (e.g., the primer pairs provided herein). In someembodiments, the presence of a hops latent viroid (HpLVd) in a cannabisplant may be determined according to one or more amplification productsgenerated using one or more primer pairs that specifically amplifysubsequences of a hops latent viroid (HpLVd) (e.g., primer pairsprovided herein). In some embodiments, the presence of a hops latentviroid (HpLVd) in a Cannabis plant may be determined according to two ormore amplification products generated using two or more primer pairsthat specifically amplify subsequences of a hops latent viroid (HpLVd)(e.g., primer pairs provided herein). In some embodiments, the presenceof a hops latent viroid (HpLVd) in a Cannabis plant may be determinedaccording to three or more amplification products generated using threeor more primer pairs that specifically amplify subsequences of a hopslatent viroid (HpLVd) (e.g., primer pairs provided herein). In someembodiments, the presence of a hops latent viroid (HpLVd) in a cannabisplant may be determined according to four or more amplification productsgenerated using four or more primer pairs that specifically amplifysubsequences of a hops latent viroid (HpLVd) (e.g., primer pairsprovided herein). In some embodiments, the presence of a hops latentviroid (HpLVd) in a Cannabis plant may be determined according to fiveor more amplification products generated using five or more primer pairsthat specifically amplify subsequences of a hops latent viroid (HpLVd)(e.g., primer pairs provided herein). In some embodiments, the presenceof a hops latent viroid (HpLVd) in a Cannabis plant may be determinedaccording to six or more amplification products generated using six ormore primer pairs that specifically amplify subsequences of a hopslatent viroid (HpLVd) (e.g., primer pairs provided herein). In someembodiments, the presence of a hops latent viroid (HpLVd) in a Cannabisplant may be determined according to seven or more amplificationproducts generated using seven or more primer pairs that specificallyamplify subsequences of a hops latent viroid (HpLVd) (e.g., primer pairsprovided herein). In some embodiments, the presence of a hops latentviroid (HpLVd) in a Cannabis plant may be determined according to eightor more amplification products generated using eight or more primerpairs that specifically amplify subsequences of a hops latent viroid(HpLVd) (e.g., primer pairs provided herein). In some embodiments, thepresence of a hops latent viroid (HpLVd) in a cannabis plant may bedetermined according to nine or more amplification products generatedusing nine or more primer pairs that specifically amplify subsequencesof a hops latent viroid (HpLVd) (e.g., primer pairs provided herein). Insome embodiments, the presence of a hops latent viroid (HpLVd) in aCannabis plant may be determined according to ten or more amplificationproducts generated using ten or more primer pairs that specificallyamplify subsequences of a hops latent viroid (HpLVd) (e.g., primer pairsprovided herein).

In some embodiments, analyzing comprises detecting one or more genotypesin a hops latent viroid. A genotype generally refers to a part of thegenetic information of an organism or pathogen (e.g., virus or viroid),which may determine one or more of its characteristics or traits(phenotypes). A genotype of a virus or viroid may refer to a particularmutation or a combination of mutations, a genetic variation or acombination or genetic variations, and/or an allele or a combination ofalleles. A genotype may specify whether an organism or viroid has areference allele or an alternate allele at a particular locus. In someembodiments, analyzing comprises detecting a genetic variation in a hopslatent viroid genome. A genotype for a hops latent viroid may specify areference allele for a particular locus in the hops latent viroidgenome. A reference allele may refer to a nucleotide present at aparticular position as provided in SEQ ID NO:1. A genotype for a hopslatent viroid may specify an alternate allele for a particular locus inthe hops latent viroid genome. An alternate allele may refer to avariant nucleotide present at a particular position in SEQ ID NO:1(i.e., a nucleotide that is different from the nucleotide at thatposition in SEQ ID NO:1).

Any suitable method for genotype assessment may be used for detecting agenetic variation in a hops latent viroid genome, such as, for example,nucleic acid sequencing (examples of which are described herein) and/ora high resolution melting (HRM) assay described herein. Generally, asequencing process and/or an HRM assay are performed in conjunction witha nucleic acid amplification method described herein (e.g., using theamplification primers provided herein). In some embodiments, one or moregenetic variations may be determined according to the presence and/orabsence of amplification products generated using certain amplificationprimers provided herein. Such primers are distinct from the primersdescribed above (i.e., primary primers, first set of primers,thermomutant-resistant primers) and may be referred to as furtherprimers, secondary primers, a second set of primers,thermomutant-specific, and/or thermomutant-sensitive primers. Forexample, certain amplification primers provided herein hybridize tosubsequences of the hops latent viroid genome that contain variantpositions (e.g., thermomutant-specific primers). The presence of avariant nucleotide in the hops latent viroid genome can result in thefailure of a thermomutant-specific primer to hybridize to itscorresponding HpLVd subsequence carrying the variant nucleotide. Suchhybridization failure results in an absence of certain amplificationproduct or products, and the absence of a certain amplification productor products can be indicative of the presence of at least one geneticvariation in the HpLVd subsequence. Examples of further primers,secondary primers, a second set of primers, thermomutant-specific,and/or thermomutant-sensitive primers are provided in Table 1 (primerslabeled tm-specific). The reverse complement for each primer also iscontemplated herein.

In some embodiments, detecting one or more genetic variations in thehops latent viroid comprises contacting the nucleic acid of the plantsample with one or more further polynucleotide primers (e.g., primersdistinct from the first set of primers described above). The nucleicacid of the plant sample may be contacted with the furtherpolynucleotide primer(s) under amplification conditions. Theamplification conditions may be the same amplification conditions asdescribed above for the first set of primers, or may be a differentamplification conditions. The amplification reaction may be the sameamplification reaction as described above for the first set of primers,or may be a different amplification reaction. In some embodiments, oneamplification reaction is performed using a combination of primers fromthe first set and primers from the second set. In some embodiments,certain forward primers from the first set pair with certain reverseprimers from the second set, and vice versa (see, e.g., Table 1 andTable 2).

In some embodiments, the further polynucleotide primers hybridize tosubsequences of SEQ ID NO:1 (i.e., subsequences of the HpLVd genome thathave not been mutated (e.g., subsequences containing nothermomutations)). Generally, the further polynucleotide primershybridize to subsequences of SEQ ID NO:1 if the subsequences are presentin the nucleic acid of a plant sample (e.g., when the plant has beeninfected with HpLVd, and where the variant positions in the subsequencescontain no mutations). Furthermore, the further polynucleotide primersdo not hybridize to subsequences of SEQ ID NO:1 if the subsequences arenot present in the nucleic acid of a plant sample (e.g., when the planthas not been infected with HpLVd, or when an HpLVd subsequence containsa mutation). In some embodiments, when a plurality of furtherpolynucleotide primers is used, a majority of the polynucleotide furtherpolynucleotide primers hybridize to subsequences of SEQ ID NO:1. Amajority of the further polynucleotide primers may refer to greater than50% of the further primers. For example, a majority of the furtherpolynucleotide primers may refer to greater than 60% of the furtherprimers, greater than 70% of the further primers, greater than 80% ofthe further primers, or greater than 90% of the further primers. In someembodiments, all (e.g., 100%) of the further polynucleotide primershybridize to subsequences of SEQ ID NO:1 (i.e., subsequences of theHpLVd genome that have not been mutated (e.g., subsequences containingno thermomutations)).

In some embodiments, the subsequences of SEQ ID NO:1 to which thefurther polynucleotide primers hybridize (also referred to as furtherprimer hybridization sites) contain one or more variant nucleotidepositions. As noted above, a variant nucleotide position refers to anucleotide (or base) at a given position in SEQ ID NO:1 that may bemutated (e.g., during thermotherapy) and/or differs among various HpLVdstrains (e.g., may contain a reference allele or an alternate allele). Asubsequence containing one or more variant positions refers to asubsequence where at least one base is subject to mutation (e.g., duringthermotherapy) and/or has at least one known alternative variant (i.e.,a known nucleotide substitution, insertion, or deletion at the variantposition).

In some embodiments, each further polynucleotide primer comprises asequence that is non-identical to any subsequence, or complementthereof, in a cannabis genome (e.g., a CS10 Cannabis genome, and/or anycannabis genome described herein). As noted above, a sequence that isnon-identical to any subsequence, or complement thereof, in a cannabisgenome generally refers to a sequence comprising one or more mismatchednucleotides when compared to any subsequence, or complement thereof, ina cannabis genome (e.g., CS10 Cannabis genome). In some embodiments,each further polynucleotide primer comprises a sequence comprising atleast two, three, four, five, six, seven, eight, nine, or ten mismatcheswhen compared to any subsequence, or complement thereof, in a cannabisgenome (e.g., CS10 Cannabis genome). In some embodiments, each furtherpolynucleotide primer comprises a sequence comprising at least sixmismatches when compared to any subsequence, or complement thereof, in acannabis genome (e.g., CS10 Cannabis genome).

The further primers provided herein (i.e., thermomutant-specificprimers) generally share a high degree of sequence identity to asubsequence, or complement thereof, of SEQ ID NO:1. In some embodiments,each further primer comprises a sequence that is at least about 90%identical to a subsequence, or complement thereof, of SEQ ID NO:1. Insome embodiments, each further primer comprises a sequence that is atleast about 95% identical to a subsequence, or complement thereof, ofSEQ ID NO:1. In some embodiments, each further primer comprises asequence that is 100% identical to a subsequence, or complement thereof,of SEQ ID NO:1.

In some embodiments, one or more further polynucleotide primers (i.e.,one or more thermomutant-specific primers) independently are chosen froma polynucleotide comprising a sequence that is at least about 90%identical to CTACGTGACTTACCTGTATGGTGGC (SEQ ID NO:2),GTGAAGAAGGAGCCGTTCCA (SEQ ID NO:7), AGAGTTGTATTCACCGGGTAGTTTCC (SEQ IDNO:14), and GCACTTTTTATGTGAACTTCTGC (SEQ ID NO:15). In some embodiments,one or more further polynucleotide primers (i.e., one or morethermomutant-specific primers) independently are chosen from apolynucleotide comprising a sequence that is at least about 95%identical to CTACGTGACTTACCTGTATGGTGGC (SEQ ID NO:2),GTGAAGAAGGAGCCGTTCCA (SEQ ID NO:7), AGAGTTGTATTCACCGGGTAGTTTCC (SEQ IDNO:14), and GCACTTTTTATGTGAACTTCTGC (SEQ ID NO:15). In some embodiments,one or more further polynucleotide primers (i.e., one or morethermomutant-specific primers) independently are chosen from apolynucleotide comprising a sequence that is 100% identical toCTACGTGACTTACCTGTATGGTGGC (SEQ ID NO:2), GTGAAGAAGGAGCCGTTCCA (SEQ IDNO:7), AGAGTTGTATTCACCGGGTAGTTTCC (SEQ ID NO:14), andGCACTTTTTATGTGAACTTCTGC (SEQ ID NO:15). In some embodiments, one or morefurther polynucleotide primers (i.e., one or more thermomutant-specificprimers) comprise CTACGTGACTTACCTGTATGGTGGC (SEQ ID NO:2),GTGAAGAAGGAGCCGTTCCA (SEQ ID NO:7), AGAGTTGTATTCACCGGGTAGTTTCC (SEQ IDNO:14), and GCACTTTTTATGTGAACTTCTGC (SEQ ID NO:15). In some embodiments,one or more further polynucleotide primers (i.e., one or morethermomutant-specific primers) consist of CTACGTGACTTACCTGTATGGTGGC (SEQID NO:2), GTGAAGAAGGAGCCGTTCCA (SEQ ID NO:7), AGAGTTGTATTCACCGGGTAGTTTCC(SEQ ID NO:14), and GCACTTTTTATGTGAACTTCTGC (SEQ ID NO:15).

In some embodiments, a primer provided herein (e.g., a further primerherein) comprises a polynucleotide where one or more nucleotidepositions contain a nonstandard nucleotide and/or a degeneratenucleotide. A nonstandard nucleotide may be, for example, a non-naturalbase, a modified base, or a universal base. A universal base is a basecapable of indiscriminately base pairing with each of the four standardnucleotide bases: A, C, G and T. Universal bases that may beincorporated into a primer herein include, but are not limited to,inosine, deoxyinosine, 2′-deoxyinosine (dl, dlnosine), nitroindole,5-nitroindole, and 3-nitropyrrole (e.g., 5′ nitroindole, deoxyinosine,deoxynebularine). A degenerate nucleotide typically refers to a mixtureof nucleotides at a given position and may be represented by a letterother than A, T, G or C. For example, a degenerate nucleotide may berepresented by R (A or G), Y (C or T), S (G or C), W (A or T), K (G orT), M (A or C), B (C or G or T), D (A or G or T), H (A or C or T), V (Aor C or G), or N (any base), for example. Such symbols for degeneratenucleotides are part of the International Union of Pure and AppliedChemistry (IUPAC) standard nomenclature for nucleotide base sequencenames and represent degenerate or nonstandard nucleotides that can bindmultiple nucleotides. For example, an “M” in a primer or probe wouldinclude a mixture of A and C at that position, and thus could bind toeither T or G in a complementary DNA strand. An “N” in a primer or probewould include a mixture of A, T, G and C at that position, and thuscould bind to any nucleotide at that position in the complementary DNAstrand.

In some embodiments, analyzing comprises detecting one or more geneticvariations in a hops latent viroid genome. In some embodiments,analyzing comprises detecting two or more genetic variations in a hopslatent viroid genome. In some embodiments, analyzing comprises detectingthree or more genetic variations in a hops latent viroid genome. In someembodiments, analyzing comprises detecting four or more geneticvariations in a hops latent viroid genome. In some embodiments,analyzing comprises detecting five or more genetic variations in a hopslatent viroid genome. In some embodiments, analyzing comprises detectingsix or more genetic variations in a hops latent viroid genome. In someembodiments, analyzing comprises detecting seven or more geneticvariations in a hops latent viroid genome. In some embodiments,analyzing comprises detecting eight or more genetic variations in a hopslatent viroid genome. In some embodiments, analyzing comprises detectingnine or more genetic variations in a hops latent viroid genome. In someembodiments, analyzing comprises detecting ten or more geneticvariations in a hops latent viroid genome.

A genetic variation may refer to a nucleotide insertion, a nucleotidedeletion, or a nucleotide substitution. An example of a nucleotidedeletion in the hops latent viroid (HpLVd) genome is a deletion of thenucleotide at position 225 of SEQ ID NO:1. A nucleotide substitution maybe referred to as a single nucleotide variation, single nucleotidemutation, or single nucleotide polymorphism (SNP). A single nucleotidevariation generally refers to a variant nucleotide at a particularposition in the HpLVd genome (SEQ ID NO:1). A variant nucleotide (alsoreferred to as a variant allele) generally refers to a nucleotide otherthan the nucleotide present at that position in SEQ ID NO:1. Forexample, position 1 of SEQ ID NO:1 is a C nucleotide, and a variantnucleotide at that position would be any nucleotide other than a Cnucleotide (e.g., A, T, or G nucleotide). Examples of single nucleotidevariations in the hops latent viroid (HpLVd) genome include a variantnucleotide at position 7 of SEQ ID NO:1, a variant nucleotide atposition 10 of SEQ ID NO:1, a variant nucleotide at position 12 of SEQID NO:1, a variant nucleotide at position 26 of SEQ ID NO:1, a variantnucleotide at position 27 of SEQ ID NO:1, a variant nucleotide atposition 28 of SEQ ID NO:1, a variant nucleotide at position 29 of SEQID NO:1, a variant nucleotide at position 30 of SEQ ID NO:1, a variantnucleotide at position 33 of SEQ ID NO:1, a variant nucleotide atposition 35 of SEQ ID NO:1, a variant nucleotide at position 43 of SEQID NO:1, a variant nucleotide at position 59 of SEQ ID NO:1, a variantnucleotide at position 121 of SEQ ID NO:1, a variant nucleotide atposition 128 of SEQ ID NO:1, a variant nucleotide at position 134 of SEQID NO:1, a variant nucleotide at position 150 of SEQ ID NO:1, a variantnucleotide at position 157 of SEQ ID NO:1, a variant nucleotide atposition 162 of SEQ ID NO:1, a variant nucleotide at position 168 of SEQID NO:1, a variant nucleotide at position 169 of SEQ ID NO:1, a variantnucleotide at position 177 of SEQ ID NO:1, a variant nucleotide atposition 200 of SEQ ID NO:1, a variant nucleotide at position 225 of SEQID NO:1, a variant nucleotide at position 229 of SEQ ID NO:1, a variantnucleotide at position 247 of SEQ ID NO:1, a variant nucleotide atposition 248 of SEQ ID NO:1, and a variant nucleotide at position 253 ofSEQ ID NO:1.

In some embodiments, a method for analyzing nucleic acid from a plantsample, comprises a) contacting nucleic acid of a plant sample with afirst set of polynucleotide primers under amplification conditions,thereby generating a first set of amplification products, where i) themajority or all of the primers in the first set of polynucleotideprimers hybridize to subsequences of SEQ ID NO:1 if present in thenucleic acid of the plant sample under the amplification conditions, ii)the subsequences of SEQ ID NO:1 to which the majority or all of theprimers in the first set of polynucleotide primers hybridize under theamplification conditions contain no variant nucleotide position, andiii) each subsequence of SEQ ID NO:1 between the subsequences to whichthe primers in the first set of polynucleotide primers hybridize containone or more variant nucleotide positions; b) contacting the nucleic acidof the plant sample with a second set of polynucleotide primers underthe amplification conditions, thereby generating a second set ofamplification products, where i) the majority or all of the primers inthe second set of polynucleotide primers hybridize to subsequences ofSEQ ID NO:1 if present in the nucleic acid of the plant sample under theamplification conditions, and ii) the subsequences of SEQ ID NO:1 towhich the majority or all of the primers in the second set ofpolynucleotide primers hybridize under the amplification conditionscontain one or more variant nucleotide positions; and c) analyzing thefirst and second sets of amplification products.

In some embodiments, analyzing comprises detecting a genetic variationsignature (e.g., a genetic variation signature for a hops latent viroidgenome). Generally, a genetic variation signature comprises genotypesdetermined at a plurality of variant nucleotide positions. A particulargenetic variation signature may comprise reference allele genotypes,alternate (i.e., variant) allele genotypes, or a combination ofreference allele genotypes and alternate (i.e., variant) allelegenotypes. Thus, a genetic variation signature may comprise acombination of variant and non-variant identities for a plurality ofnucleotide positions in a hops latent viroid genome. A genetic variationsignature in certain contexts may be referred to as a serotype, aserovar, a barcode, or a haplotype.

In some embodiments, a genetic variation signature comprises genotypesdetermined at two or more variant nucleotide positions in a hops latentviroid genome. In some embodiments, a genetic variation signaturecomprises genotypes determined at three or more variant nucleotidepositions in a hops latent viroid genome. In some embodiments, a geneticvariation signature comprises genotypes determined at four or morevariant nucleotide positions in a hops latent viroid genome. In someembodiments, a genetic variation signature comprises genotypesdetermined at five or more variant nucleotide positions in a hops latentviroid genome. In some embodiments, a genetic variation signaturecomprises genotypes determined at six or more variant nucleotidepositions in a hops latent viroid genome. In some embodiments, a geneticvariation signature comprises genotypes determined at seven or morevariant nucleotide positions in a hops latent viroid genome. In someembodiments, a genetic variation signature comprises genotypesdetermined at eight or more variant nucleotide positions in a hopslatent viroid genome. In some embodiments, a genetic variation signaturecomprises genotypes determined at nine or more variant nucleotidepositions in a hops latent viroid genome. In some embodiments, a geneticvariation signature comprises genotypes determined at ten or morevariant nucleotide positions in a hops latent viroid genome.

In some embodiments, analyzing comprises identifying a hops latentviroid trait according to one or more genetic variations in a hopslatent viroid genome. In some embodiments, analyzing comprisesidentifying a hops latent viroid trait according to a genetic variationsignature determined for a hops latent viroid genome. Identifying a hopslatent viroid trait according to one or more genetic variations and/or agenetic variation signature may be referred to as classifying agenotype; associating one or more phenotypes of an infected plant (e.g.,an infected cannabis plant) with one or more genotypes and/or geneticvariations for a pathogen (e.g., HpLVd); and/or associating one or moredisease phenotypes in a plant (e.g., a Cannabis plant) with a particularHpLVd genotype. A hops latent viroid trait (or phenotypic trait) mayrefer to any distinguishing quality or characteristic of the viroiditself and/or phenotype expressed by a plant infected by the viroid. Insome embodiments, a method comprises identifying an HpLVd trait orsegment of the HpLVd genome that is an indicator of whether a particularHpLVd variant in a particular cultivar is more or lessvirulent/symptomatic. Without being limited by theory, HpLVd RNA may becomplementary to certain genes, or fragments thereof, in the plant,which, when hybridized, may prevent the plant gene expression by actingas a silencing/interfering RNA type molecule. In some embodiments, amethod herein comprises matching genotypes of HpLVd with cannabisphenotypes and/or cannabis genotypes that either confer resistance toinfection or susceptibility to infection, such that cannabis genotypessusceptible to certain HpLVd genotypes may be identified and/or cannabisplants resistant to HpLVd may be bred.

A hops latent viroid trait may include, for example, infectiousness andor contagiousness of the viroid; presence or absence of symptoms in aninfected plant; type, pervasiveness, and/or severity of symptoms in aninfected plant; degree of recovery of an infected plant; and/orresponsiveness to treatment. Symptoms of an infected plant may include,for example, loss of vigor, stunting, abnormal stretching, reduction inyield, reduction in potency, changes in morphology, reduction or lack ofoil, small trichome heads, malformed trichomes, misshapen leaves, leavesthat are yellowish in color, brittle stems, an outwardly horizontalplant structure, and reduced flower mass and trichomes.

AMV, BCTV

A plant may be a Cannabis plant and a pathogen may be an Alfalfa MosaicVirus (AMV) or a Beet Curly Top Virus (BCTV). Accordingly, in someembodiments, analyzing comprises detecting the presence, absence and/oramount of AMV or BCTV in a Cannabis plant. In some embodiments,analyzing comprises detecting the presence, absence and/or amount ofHpLVd, AMV or BCTV or any combination thereof (e.g., HpLVd and AMV; orAMV and BCTV; or HpLVd and BCTV; or HpLVd and AMV and BCTV) in aCannabis plant. Presence of AMV or BCTV in a Cannabis plant may bedetermined according to amplification products generated using one ormore polynucleotide primer pairs that specifically amplify subsequencesof an AMV or a BCTV (e.g., the polynucleotide primer pairs providedherein). In some embodiments, the presence of AMV or BCTV in a Cannabisplant may be determined according to one or more amplification productsgenerated using one or more primer pairs that specifically amplifysubsequences of AMV or BCTV, respectively (e.g., the polynucleotideprimer pairs provided herein). In some embodiments, the presence of AMVor BCTV in a Cannabis plant may be determined according to two or moreamplification products generated using two or more primer pairs thatspecifically amplify subsequences of AMV or BCTV, respectively (e.g.,the polynucleotide primer pairs provided herein). In some embodiments,the presence of AMV or BCTV in a Cannabis plant may be determinedaccording to three or more amplification products generated using threeor more primer pairs that specifically amplify subsequences of AMV orBCTV, respectively (e.g., the polynucleotide primer pairs providedherein). In some embodiments, the presence of AMV or BCTV in a Cannabisplant may be determined according to four or more amplification productsgenerated using four or more primer pairs that specifically amplifysubsequences of AMV or BCTV, respectively (e.g., the polynucleotideprimer pairs provided herein). In some embodiments, the presence of AMVor BCTV in a Cannabis plant may be determined according to five or moreamplification products generated using five or more primer pairs thatspecifically amplify subsequences of AMV or BCTV, respectively (e.g.,the polynucleotide primer pairs provided herein). In some embodiments,the presence of AMV or BCTV in a Cannabis plant may be determinedaccording to six or more amplification products generated using six ormore primer pairs that specifically amplify subsequences of AMV or BCTV,respectively (e.g., the polynucleotide primer pairs provided herein). Insome embodiments, the presence of AMV or BCTV in a Cannabis plant may bedetermined according to seven or more amplification products generatedusing seven or more primer pairs that specifically amplify subsequencesof AMV or BCTV, respectively (e.g., the polynucleotide primer pairsprovided herein). In some embodiments, the presence of AMV or BCTV in aCannabis plant may be determined according to eight or moreamplification products generated using eight or more primer pairs thatspecifically amplify subsequences of AMV or BCTV, respectively (e.g.,the polynucleotide primer pairs provided herein). In some embodiments,the presence of AMV or BCTV in a Cannabis plant may be determinedaccording to nine or more amplification products generated using nine ormore primer pairs that specifically amplify subsequences of AMV or BCTV,respectively (e.g., the polynucleotide primer pairs provided herein). Insome embodiments, the presence of AMV or BCTV in a Cannabis plant may bedetermined according to ten or more amplification products generatedusing ten or more primer pairs that specifically amplify subsequences ofAMV or BCTV, respectively (e.g., the polynucleotide primer pairsprovided herein).

In embodiments, analyzing comprises detecting one or more variants. Avariant generally refers to a change in the sequence of the nucleic acidand/or proteins encoded by the nucleic acid, such as an insertion,deletion, or substitution (mutation). In some embodiments, analyzingcomprises detecting one or more genotypes in AMV or BCTV. A genotypegenerally refers to a part of the genetic information of an organism orpathogen (e.g., viroid), which may determine one or more of itscharacteristics or traits (phenotypes). A genotype of a virus may referto a particular mutation or a combination of mutations, a geneticvariation or a combination or genetic variations, and/or an allele or acombination of alleles. A genotype may specify whether an organism orpathogen has a reference allele or an alternate allele at a particularlocus. In some embodiments, analyzing comprises detecting a geneticvariation in one or more of HpLVd, AMV and BCTV. A genotype for HpLVdmay specify a reference allele for a particular locus in the HpLVdgenome. A reference allele may refer to a nucleotide present at aparticular position as provided in SEQ ID NO:1. A genotype for a HpLVdmay specify an alternate allele for a particular locus in the HpLVdgenome. An alternate allele may refer to a variant nucleotide present ata particular position in SEQ ID NO:1 (i.e., a nucleotide that isdifferent from the nucleotide at that position in SEQ ID NO:1).

A genotype for AMV may specify a reference allele for a particular locusin the AMV genome. A reference allele may refer to a nucleotide presentat a particular position as provided in SEQ ID NO:91. A genotype for aAMV may specify an alternate allele for a particular locus in the AMVgenome. An alternate allele may refer to a variant nucleotide present ata particular position in SEQ ID NO:91 (i.e., a nucleotide that isdifferent from the nucleotide at that position in SEQ ID NO:91). Agenotype for BCTV may specify a reference allele for a particular locusin the BCTV genome. A reference allele may refer to a nucleotide presentat a particular position as provided in one or more of SEQ ID NOS:110,112, 114, 116, 118 and 120. A genotype fora BCTV may specify analternate allele for a particular locus in the BCTV genome. An alternateallele may refer to a variant nucleotide present at a particularposition in one or more of SEQ ID NOS:110, 112, 114, 116, 118 and 120(i.e., a nucleotide that is different from the nucleotide at thecorresponding position in SEQ ID NOS:110, 112, 114, 116, 118 and 120,respectively).

Any suitable method for genotype assessment may be used for detecting agenetic variation in a genome of a pathogen, such as, for example,nucleic acid sequencing (examples of which are described herein) and/ora high resolution melting (HRM) assay described herein. Generally, asequencing process and/or an HRM assay are performed in conjunction witha nucleic acid amplification method described herein (e.g., using theamplification primers provided herein). In some embodiments, one or moregenetic variations may be determined according to the presence and/orabsence of amplification products generated using certain amplificationprimers provided herein.

Also provided herein, in certain aspects, are multiplexed methods ofdetermining the presence, absence and/or amount of one or more pathogensin one or more plant cultivars. In certain aspects, the multiplexedmethod comprises one or more of:

-   -   (1) determining the presence, absence and/or amount of more than        one non-overlapping amplicon of a pathogen that may have        infected a plant cultivar;    -   (2) determining the presence, absence and/or amount of more than        one pathogen that may have infected a plant cultivar by        determining the presence, absence and/or amount of one or more        amplicons of each pathogen;    -   (3) determining the presence, absence and/or amount of one or        more pathogens in a plurality of plant cultivars;    -   (4) quantifying an amplicon of a pathogen using more than one        non-overlapping polynucleotide probe.

Any of the plant pathogens described herein and known to those of skillin the art can be analyzed in the multiplexed methods provided herein.In embodiments, the multiplexed methods provided herein can be used toanalyze more than one pathogen, where the one or more, two or more orthree or more pathogens analyzed are selected from among Hops LatentViroid (HpLVd), Alfalfa Mosaic Virus (AMV), Beet Curly Top Virus (BCTV),Hemp Streak Virus (HSV), Hemp Mosaic Virus (HMV), Tomato spotted wiltvirus (TSWV), Sunn-Hemp Mosaic Virus (SHMV), Arabis Mosaic Virus (ArMV),Cucumber Mosaic Virus (CMV), Lettuce Chlorosis Virus (LCV), TobaccoRingspot Virus (TRSV), Tomato Ringspot Virus (TomRSV), and TobaccoStreak Virus (TSV), Cannabis Cryptic Virus (CCV), Potato Spindle TubularViroid (PSTV), Coconut cadang cadang viroid (CCCV), Apple scar skinviroid (ASSV), Avocado sunblotch viroid (ASBV), Tobacco streak virus(TSV), Tomato mosaic virus (ToMV), Euonymous Ringspot Virus (ERSV), ElmMosaic Virus (EMV), and Hops Stunting Virus (HpSV). In certainembodiments, the multiplexed methods provided herein can be used toanalyze more than one pathogen, where one or more, two or more or threeor more pathogens analyzed are selected from among Hops Latent Viroid(HpLVd), Alfalfa Mosaic Virus (AMV) and Beet Curly Top Virus (BCTV). Inany of the multiplexed methods provided herein, a positive controlamplicon can be generated using a polynucleotide primer pair that iscapable of specifically hybridizing to and amplifying a subsequence ofthe nucleic acid of the plant genome, or to a complement thereof,wherein the subsequence of the nucleic acid of the plant genome, or thecomplement thereof, is non-identical to any subsequence of the nucleicacid of the pathogen, or to any complement thereof; and determining thepresence, absence and/or amount of at least one amplicon that is anamplification product of the polynucleotide primer pair that is capableof specifically hybridizing to and amplifying a subsequence of thenucleic acid of the plant genome, thereby determining whether theamplification conditions are effective for generating amplicons. Inembodiments, the subsequence of the nucleic acid of the plant genomecomprises all or part of a gene selected from among 26S rRNA,beta-tubulin, ATP Synthase, an rRNA subunit, glyceraldehyde-3-phosphatedehydrogenase, Ubiquitin-conjugating enzyme E2, eukaryotic transcriptionfactors, eukaryotic initiation factor 1 and beta-actin. In certainembodiments, the subsequence of the nucleic acid of the plant genomecomprises all or part of the 26SrRNA gene.

Examples of configurations of a multiplexed method are provided below.These examples depict various combinations for determining the presence,absence and/or amount of one or more pathogens selected from amongHpLVd, AMV and BCTV, with or without and an internal (plant genomespecific) positive control (IPC), with each amplicon and/orpolynucleotide probe uniquely labeled, such as with a unique fluorescentlabel.

Multiplex 1:

-   HpLVd: B-fwd (SEQ ID NO:4) with F-rev (SEQ ID NO:12) using Probe 1,    2, 3, 4, or 5 (SEQ ID NOS:16-20)-   AMV: A-fwd (SEQ ID NO:80) with A-rev (SEQ ID NO:81) with Probe A    (SEQ ID NO:87)-   BCTV: PE_RE_fwd (SEQ ID NO:102) with PE_RE_Rev (SEQ ID NO:103) and    PE_RE_Probe (SEQ ID NO:104)-   IPC: 26S rRNA-fwd (SEQ ID NO:107) with 26S rRNA-rev (SEQ ID NO:108)    and 26S rRNA Probe (SEQ ID NO:109)

Multiplex 2:

-   HpLVd: C-fwd (SEQ ID NO:6) with E-Rev (SEQ ID NO:10) using Probe 1,    3, or 5 (SEQ ID NOS: 16, 18 and 20, respectively)-   AMV: B-fwd (SEQ ID NO:82) with B-rev (SEQ ID NO:83) with Probe B    (SEQ ID NO:89)-   BCTV: RCRI_CCR_Fwd (SEQ ID NO:99) with Degen_RCRI_CCR_Rev (SEQ ID    NO:100) with RCRI_CCR_Probe (SEQ ID NO:101)-   IPC: 26S rRNA-fwd (SEQ ID NO:107) with 26S rRNA-rev (SEQ ID NO:108)    and 26S rRNA Probe (SEQ ID NO:109)

Multiplex 3:

-   HpLVd: D-fwd (SEQ ID NO:9) with D-rev (SEQ ID NO:8) using Probe 1,    3, or 5 (SEQ ID NOS: 16, 18 and 20, respectively)-   AMV: A-fwd (SEQ ID NO:80) with C-rev (SEQ ID NO:84) with Probe A    (SEQ ID NO:87) or B (SEQ ID NO:89)-   BCTV: DRP_MP_Fwd (SEQ ID NO:93) with DRP_MP_Rev (SEQ ID NO:94) using    DRP_MP_Probe (SEQ ID NO:95)-   IPC: 26S rRNA-fwd (SEQ ID NO:107) with 26S rRNA-rev (SEQ ID NO:108)    and 26S rRNA Probe (SEQ ID NO:109)

In embodiments, a single pathogen can be analyzed in a multiplexedformat using more than one set of polynucleotide primer pairs. Examplesof this configuration are depicted below:

Multiplex 4 (Pathogen—HpLVd):

-   B-fwd (SEQ ID NO:4) with F-rev (SEQ ID NO:12) using Probe 1, 2, 3,    4, or 5 (SEQ ID NOS:16-20)-   E-fwd (SEQ ID NO:11) with E-Rev (SEQ ID NO:10) using Probe 3 or 5    (SEQ ID NO:18 or 20, respectively)-   D-fwd (SEQ ID NO:9) with D-rev (SEQ ID NO:8) using Probe 1, 3, or 5    (SEQ ID NOS: 16, 18 and 20, respectively)-   IPC: 26S rRNA-fwd (SEQ ID NO:107) with 26S rRNA-rev (SEQ ID NO:108)    and 26S rRNA Probe (SEQ ID NO:109)

Multiplex 5 (Pathogen—HpLVd):

-   F-fwd (SEQ ID NO:13) with F-rev (SEQ ID NO:12) using Probe 3 or 5    (SEQ ID NO:18 or 20, respectively)-   D-fwd (SEQ ID NO:9) with D-rev (SEQ ID NO:8) using Probe 1, 3, or 5    (SEQ ID NOS: 16, 18 and 20, respectively)-   B-fwd (SEQ ID NO:4) with B-rev (SEQ ID NO:5) using Probe 2 or 4 (SEQ    ID NO:17 or 19, respectively)

Multiplex 6 (Pathogen—AMV):

-   A-fwd (SEQ ID NO:80) with A-rev (SEQ ID NO:81) with Probe A (SEQ ID    NO:87)-   B-fwd (SEQ ID NO:82) with B-rev (SEQ ID NO:83) with Probe B (SEQ ID    NO:89)-   IPC: 26S rRNA-fwd (SEQ ID NO:107) with 26S rRNA-rev (SEQ ID NO:108)    and 26S rRNA Probe (SEQ ID NO:109)    Multiplex 7 (Pathogen—BCTV): (DNA virus, therefore, could be run on    cDNA as RT-qPCR multiplex or on gDNA (genomic DNA) as qPCR    multiplex)-   PE_RE_fwd (SEQ ID NO:102) with PE_RE_Rev (SEQ ID NO:103) and    PE_RE_Probe (SEQ ID NO:104)-   RCRI_CCR_Fwd (SEQ ID NO:99) with Degen_RCRI_CCR_Rev (SEQ ID NO:100)    with RCRI_CCR_Probe (SEQ ID NO:101)-   DRP_MP_Fwd (SEQ ID NO:93) with DRP_MP_Rev (SEQ ID NO:94) using    DRP_MP_Probe (SEQ ID NO:95)-   IPC: 26S rRNA-fwd (SEQ ID NO:107) with 26S rRNA-rev (SEQ ID NO:108)    and 26S rRNA Probe (SEQ ID NO:109)

In certain embodiments, more than one pathogen can be analyzed in amultiplexed format using more than one set of polynucleotide primerpairs targeting unique regions with uniquely labeled probes as depictedbelow. In embodiments, an IPC may not be analyzed in the multiplex.

Multiplex 8 (Pathogens—BCTV and HpLVd):

-   BCTV-1: PE_RE_fwd (SEQ ID NO:102) with PE_RE_Rev (SEQ ID NO:103) and    PE_RE_Probe (SEQ ID NO:104)-   BCTV-2: RCRI_CCR_Fwd (SEQ ID NO:99) with Degen_RCRI_CCR_Rev (SEQ ID    NO:100) with RCRI_CCR_Probe (SEQ ID NO:101)-   HpLVd-1: B-fwd (SEQ ID NO:4) with F-rev (SEQ ID NO:12) using Probe    1, 2, 3, 4, or 5 (SEQ ID NOS:16-20)-   HpLVd-2: D-fwd (SEQ ID NO:9) with D-rev (SEQ ID NO:8) using Probe 1,    3, or 5 (SEQ ID NOS: 16, 18 and 20, respectively)

Multiplex 9 (Pathogens—BCTV and AMV):

-   BCTV-1: PE_RE_fwd (SEQ ID NO:102) with PE_RE_Rev (SEQ ID NO:103) and    PE_RE_Probe (SEQ ID NO:104)-   BCTV-2: RCRI_CCR_Fwd (SEQ ID NO:99) with Degen_RCRI_CCR_Rev (SEQ ID    NO:100) with RCRI_CCR_Probe (SEQ ID NO:101)-   AMV-1: A-fwd (SEQ ID NO:80) with A-rev (SEQ ID NO:81) with Probe A    (SEQ ID NO:87)-   AMV-2: B-fwd (SEQ ID NO:82) with B-rev (SEQ ID NO:83) with Probe B    (SEQ ID NO:89)

Multiplex 10 (Pathogens—HpLVd and AMV):

-   HpLVd-1: B-fwd (SEQ ID NO:4) with F-rev (SEQ ID NO:12) using Probe    1, 2, 3, 4, or 5 (SEQ ID NOS:16-20)-   HpLVd-2: D-fwd (SEQ ID NO:9) with D-rev (SEQ ID NO:8) using Probe 1,    3, or 5 (SEQ ID NOS: 16, 18 and 20, respectively)-   AMV-1: A-fwd (SEQ ID NO:80) with A-rev (SEQ ID NO:81) with Probe A    (SEQ ID NO:87)-   AMV-2: B-fwd (SEQ ID NO:82) with B-rev (SEQ ID NO:83) with Probe B    (SEQ ID NO:89)

In certain embodiments, the multiplexed methods provided herein includeamplifying more than one non-overlapping subsequences of the genome of apathogen, thereby generating more than one amplicon and providingadditional verification regarding the presence, absence and/or amount ofthe pathogen. Differences in Cq values for each of the amplicons mayprovide information regarding the presence of a variant of the pathogenand/or the presence of a change in genotype when compared to the nucleicacid and/or genotype of the wild-type pathogen. Examples of such“multi-amplicon” multiplex reactions are depicted below:

Multiplex 11 (Pathogen—AMV):

-   AMV-A-Fwd (SEQ ID NO:80) with AMV—C-Rev (SEQ ID NO:84) using Probe    A-degen (SEQ ID NO:88)-   AMV-D-Fwd (SEQ ID NO:85) with AMV-D-Rev (SEQ ID NO:86) using Probe D    (SEQ ID NO:90) OR-   AMV-A-Fwd (SEQ ID NO:80) with AMV—B-Rev (SEQ ID NO:83) using Probe B    (SEQ ID NO:89)-   AMV-D-Fwd (SEQ ID NO:85) with AMV-D-Rev (SEQ ID NO:86) using Probe D    (SEQ ID NO:90)

Multiplex 12 (Pathogen—BCTV):

-   Fwd_PE_RCRI (SEQ ID NO:96) with Degen_Rev_PE_RCRI (SEQ ID NO:97)    using Degen_Probe_PE_RCRI (SEQ ID NO:98)-   Fwd_RCRI_CCR (SEQ ID NO:99) with Degen_Rev_RCRI_CCR (SEQ ID NO:100)    using Probe_RCRI_CCR (SEQ ID NO:101)

In certain embodiments, the multiplexed methods provided herein includeusing more than one non-overlapping polynucleotide probe to quantitate asingle amplicon of a plant pathogen. In embodiments, the relative Cqvalues for each polynucleotide probe can indicate whether or not genomicvariations (insertions, deletions, mutations) are present within theamplicon. Examples of such multiplex reactions are depicted below:

Multiplex 13 (Pathogen—BCTV):

-   Fwd_DRP_MP (SEQ ID NO:93) with Rev-2_DRP_MP_Degen (SEQ ID NO:105)    using Probe-2_DRP_MP _Degen (SEQ ID NO:106)-   DRP_MP_Fwd (SEQ ID NO:93) with DRP_MP_Rev (SEQ ID NO:94) using    DRP_MP_Probe (SEQ ID NO:95)

Multiplex 14 (Pathogen—AMV):

-   AMV-A-Fwd (SEQ ID NO:80) with AMV—C-Rev (SEQ ID NO:84) using Probe    A-degen (SEQ ID NO:88) & Probe B (SEQ ID NO:89)-   OR-   AMV-A-Fwd (SEQ ID NO:80) with AMV—B-Rev (SEQ ID NO:83) using Probe    A-degen (SEQ ID NO:88) & Probe B (SEQ ID NO:89)

In certain embodiments, when the pathogen is HpLVd, the multiplexedmethods provided herein can determine the extent of mutation in thegenome of the viroid (e.g., due to heating) by comparing Cq values of apolynucleotide probe used to quantify an amplicon obtained using athermomutant specific pair of polynucleotide primers and apolynucleotide probe used to quantify an amplicon obtained using athermomutant resistant pair of polynucleotide primers. Examples of suchmultiplexing reactions are depicted below:

Multiplex 15 (Pathogen—HpLVd):

-   HpLVd_1-Fwd (SEQ ID NO:77) with HpLVd_1-rev (SEQ ID NO:78) using    HpLVd_1 Probe (SEQ ID NO:79) (Thermomutant-Specific)-   F-Fwd (SEQ ID NO:13) with F-Rev (SEQ ID NO:12) using Probes 3 and/or    5 (SEQ ID NOS:18 and/or 20, respectively) (Thermomutant-Resistant)-   OR-   HpLVd_1-Fwd (SEQ ID NO:77) with HpLVd_1-rev (SEQ ID NO:78) using    HpLVd_1 Probe (SEQ ID NO:79) (Thermomutant-Specific)-   E-Fwd (SEQ ID NO:11) with E-Rev (SEQ ID NO:10) using Probes 3 and/or    5 (SEQ ID NOS:18 and/or 20, respectively) (Thermomutant-Resistant)

Multiplex 16 (Pathogen—HpLVd):

-   E-Fwd (SEQ ID NO:11) with E-Rev (SEQ ID NO:10) using Probes 3 and 5    (SEQ ID NOS:18 and 20, respectively) (Thermomutant-Resistant)-   OR-   F-Fwd (SEQ ID NO:13) with F-Rev (SEQ ID NO:12) using Probes 3 and 5    (SEQ ID NOS:18 and 20, respectively) (Thermomutant-Resistant)-   OR (for Triplicate Verification)-   B-Fwd (SEQ ID NO:4) with F-rev (SEQ ID NO:12) using Probe    combinations (2, 3, & 5 (SEQ ID NOS:17, 18 and 20, respectively) OR    1, 4, & 5 (SEQ ID NOS:16, 19 and 20, respectively)).

Samples

Provided herein are methods and compositions for processing, preparing,and/or analyzing nucleic acid. Nucleic acid or a nucleic acid mixtureutilized in methods and compositions described herein may be isolatedfrom a sample (e.g., a test sample) obtained from a plant. A plant canbe any plant capable of being infected by a hops latent viroid (HpLVd)(e.g., Humulus lupulus (hop) plant, Cannabis plant). A plant can be anyplant capable of being infected by a plant pathogen. A plant can be anyplant capable of being infected by one or more pathogen (plant virus)selected from among Hops Latent Viroid (HpLVd), Alfalfa Mosaic Virus(AMV), Beet Curly Top Virus (BCTV), Hemp Streak Virus (HSV), Hemp MosaicVirus (HMV), Tomato spotted wilt virus (TSWV), Sunn-Hemp Mosaic Virus(SHMV), Arabis Mosaic Virus (ArMV), Cucumber Mosaic Virus (CMV), LettuceChlorosis Virus (LCV), Tobacco Ringspot Virus (TRSV), Tomato RingspotVirus (TomRSV), and Tobacco Streak Virus (TSV), Cannabis Cryptic Virus(CCV), Potato Spindle Tubular Viroid (PSTV), Coconut cadang cadangviroid (CCCV), Apple scar skin viroid (ASSV), Avocado sunblotch viroid(ASBV), Tobacco streak virus (TSV), Tomato mosaic virus (ToMV),Euonymous Ringspot Virus (ERSV), Elm Mosaic Virus (EMV), and HopsStunting Virus (HpSV).

The term Cannabis generally refers to a genus of flowering plants in thefamily Cannabaceae, which contains at least 3 species: Cannabis sativa,Cannabis indica, and Cannabis ruderalis. A plant may be a plant infectedwith HpLVd or other plant pathogen, a plant suspected of being infectedwith HpLVd or other plant pathogen, a plant treated for an HpLVd orother pathogenic infection (e.g., heat treated), a plant recovering froman HpLVd or other pathogenic infection, a plant with a history of HpLVdinfections, a plant obtaining an HpLVd or other pathogenic screen, aplant sharing a cultivation space with another plant infected with HpLVdor other plant pathogen, a plant grown in a cultivation space with ahistory of HpLVd or other pathogenic infections, a plant derived from aplant infected with HpLVd (e.g., derived from a cutting of a plantinfected with HpLVd) or other plant pathogens, a plant subjected to acleaning process, and/or a cutting or explant thereof. The term cleaninggenerally refers to a process of removing one or more contaminants froma plant. If the contaminant is a pathogen (e.g., HpLVd, AMV, BCTV),example methods include one or more of thermotherapy of meristems,chemotherapy, meristem-tip culture, and use of chemicals in a media.

In some embodiments, a plant may be a cutting or explant of a wholeplant. The term cutting generally refers to a section of a plant that isthe starting material for vegetative propagation (i.e., asexual plantreproduction). The term explant, with reference to plant tissue culture,generally refers to living plant tissue that is removed from the naturalsite of growth and placed in sterile medium for culture. This can be ofany tissue type such as leaves, roots, stems, or any portion taken froma plant and used to initiate tissue culture.

A nucleic acid sample may be isolated or obtained from any type ofsuitable biological (i.e., plant) specimen or sample (e.g., a testsample). A nucleic acid sample may be isolated or obtained from a singleplant cell, a plurality of plant cells (e.g., cultured plant cells),plant cell culture media, conditioned plant cell culture media, or planttissue (e.g., leaves, roots, stems).

A sample may be heterogeneous. For example, a sample may include morethan one cell type and/or one or more nucleic acid species. In someinstances, a sample may include host plant nucleic acid and pathogennucleic acid. In some instances, a sample may include nucleic acid froma Cannabis genome and nucleic acid from the genome of a plant pathogen,such as an HpLVd, AMV or BCTV genome. In some instances, a sample mayinclude a minority nucleic acid species and a majority nucleic acidspecies. In some instances, a sample may include plant cells and/ornucleic acid from a single plant or may include plant cells and/ornucleic acid from multiple plants.

Nucleic Acid

Provided herein are methods and compositions for processing, preparing,and/or analyzing nucleic acid. The terms nucleic acid(s), nucleic acidmolecule(s), nucleic acid fragment(s), target nucleic acid(s), nucleicacid template(s), template nucleic acid(s), nucleic acid target(s),target nucleic acid(s), polynucleotide(s), polynucleotide fragment(s),target polynucleotide(s), polynucleotide target(s), and the like may beused interchangeably throughout the disclosure. The terms refer tonucleic acids of any composition from, such as DNA (e.g., complementaryDNA (cDNA; synthesized from any RNA or DNA of interest), genomic DNA(gDNA), genomic DNA fragments, mitochondrial DNA (mtDNA), recombinantDNA (e.g., plasmid DNA), and the like), RNA (e.g., message RNA (mRNA),short inhibitory RNA (siRNA), ribosomal RNA (rRNA), transfer RNA (tRNA),microRNA, transacting small interfering RNA (ta-siRNA), natural smallinterfering RNA (nat-siRNA), small nucleolar RNA (snoRNA), small nuclearRNA (snRNA), long non-coding RNA (IncRNA), non-coding RNA (ncRNA),transfer-messenger RNA (tmRNA), precursor messenger RNA (pre-mRNA),small Cajal body-specific RNA (scaRNA), piwi-interacting RNA (piRNA),endoribonuclease-prepared siRNA (esiRNA), small temporal RNA (stRNA),signal recognition RNA, telomere RNA, and the like), and/or DNA or RNAanalogs (e.g., containing base analogs, sugar analogs and/or anon-native backbone and the like), RNA/DNA hybrids and polyamide nucleicacids (PNAs), all of which can be in single- or double-stranded form,and unless otherwise limited, can encompass known analogs of naturalnucleotides that can function in a similar manner as naturally occurringnucleotides. A nucleic acid may be, or may be from, a plant, a viroid, aplasmid, autonomously replicating sequence (ARS), mitochondria,centromere, artificial chromosome, chromosome, or other nucleic acidable to replicate or be replicated in vitro or in a host cell, a cell, acell nucleus or cytoplasm of a cell in certain embodiments. A templatenucleic acid in some embodiments can be from a single chromosome (e.g.,a nucleic acid sample may be from one chromosome of a sample obtainedfrom a diploid organism). Unless specifically limited, the termencompasses nucleic acids containing known analogs of naturalnucleotides that have similar binding properties as the referencenucleic acid and are metabolized in a manner similar to naturallyoccurring nucleotides. Unless otherwise indicated, a particular nucleicacid sequence also implicitly encompasses conservatively modifiedvariants thereof (e.g., degenerate codon substitutions), alleles,orthologs, single nucleotide polymorphisms (SNPs), and complementarysequences as well as the sequence explicitly indicated. Specifically,degenerate codon substitutions may be achieved by generating sequencesin which the third position of one or more selected (or all) codons issubstituted with mixed-base and/or deoxyinosine residues. The termnucleic acid may be used interchangeably with locus, gene, cDNA, andmRNA encoded by a gene. The term also may include, as equivalents,derivatives, variants and analogs of RNA or DNA synthesized fromnucleotide analogs, single-stranded (“sense” or “antisense,” “plus”strand or “minus” strand, “forward” reading frame or “reverse” readingframe) and double-stranded polynucleotides. The term “gene” refers to asection of DNA involved in producing a polypeptide chain; and generallyincludes regions preceding and following the coding region (leader andtrailer) involved in the transcription/translation of the gene productand the regulation of the transcription/translation, as well asintervening sequences (introns) between individual coding regions(exons). A nucleotide or base generally refers to the purine andpyrimidine molecular units of nucleic acid (e.g., adenine (A), thymine(T), guanine (G), and cytosine (C)). For RNA, the base thymine isreplaced with uracil. Nucleic acid length or size may be expressed as anumber of bases.

Target nucleic acids may be any nucleic acids of interest. Nucleic acidsmay be polymers of any length composed of deoxyribonucleotides (i.e.,DNA bases), ribonucleotides (i.e., RNA bases), or combinations thereof,e.g., 10 bases or longer, 20 bases or longer, 50 bases or longer, 100bases or longer, 200 bases or longer, 300 bases or longer, 400 bases orlonger, 500 bases or longer, 1000 bases or longer, 2000 bases or longer,3000 bases or longer, 4000 bases or longer, 5000 bases or longer. Incertain aspects, nucleic acids are polymers composed ofdeoxyribonucleotides (i.e., DNA bases), ribonucleotides (i.e., RNAbases), or combinations thereof, e.g., 10 bases or less, 20 bases orless, 50 bases or less, 100 bases or less, 200 bases or less, 300 basesor less, 400 bases or less, 500 bases or less, 1000 bases or less, 2000bases or less, 3000 bases or less, 4000 bases or less, or 5000 bases orless.

Nucleic acid may be single or double stranded. Single stranded DNA(ssDNA), for example, can be generated by denaturing double stranded DNAby heating or by treatment with alkali, for example. Accordingly, insome embodiments, ssDNA is derived from double-stranded DNA (dsDNA).

Nucleic acid (e.g., nucleic acid targets, polynucleotides, primers,polynucleotide primers, polynucleotide primer pairs, sequences, andsubsequences) may be described herein as being complementary to anothernucleic acid, hybridizing to another nucleic acid, and/or being capableof hybridizing to another nucleic acid. The terms “complementary” or“complementarity” or “hybridization” generally refer to a nucleotidesequence that base-pairs by non-covalent bonds to a region of a nucleicacid (e.g., a primer that hybridizes to a subsequence of HpLVd or otherplant pathogen, a primer that is complementary to a subsequence of HpLVdor other plant pathogen). In the canonical Watson-Crick base pairing,adenine (A) forms a base pair with thymine (T), and guanine (G) pairswith cytosine (C) in DNA. In RNA, thymine (T) is replaced by uracil (U).Thus, A is complementary to T and G is complementary to C. In RNA, A iscomplementary to U and vice versa. In a DNA-RNA duplex, A (in a DNAstrand) is complementary to U (in an RNA strand). Typically,“complementary” or “complementarity” or “hybridize” or “capable ofhybridizing” refers to a nucleotide sequence that is at least partiallycomplementary. These terms may also encompass duplexes that are fullycomplementary such that every nucleotide in one strand is complementaryor hybridizes to every nucleotide in the other strand in correspondingpositions.

In certain instances, a nucleotide sequence may be partiallycomplementary to a target, in which not all nucleotides arecomplementary to every nucleotide in the target nucleic acid in all thecorresponding positions. For example, a primer may be perfectly (i.e.,100%) complementary to an HpLVd or other plant pathogen subsequence, ora primer may share some degree of complementarity to an HpLVd or otherplant pathogen subsequence which is less than perfect (e.g., 70%, 75%,85%, 90%, 95%, 99%). In some embodiments, a primer (e.g., athermomutant-resistant primer) is 100% complementary to an HpLVdsubsequence. In some embodiments, a plurality of primers (e.g., aplurality of thermomutant-resistant primers) are 100% complementary toHpLVd subsequences.

The percent identity of two nucleotide sequences can be determined byaligning the sequences for optimal comparison purposes (e.g., gaps canbe introduced in the sequence of a first sequence for optimalalignment). The nucleotides at corresponding positions are thencompared, and the percent identity between the two sequences is afunction of the number of identical positions shared by the sequences(i.e., % identity=# of identical positions/total # of positions×100).When a position in one sequence is occupied by the same nucleotide asthe corresponding position in the other sequence, then the molecules areidentical at that position.

In some embodiments, nucleic acids in a mixture of nucleic acids areanalyzed. A mixture of nucleic acids can comprise two or more nucleicacid species having the same or different nucleotide sequences,different lengths, different origins (e.g., genomic origins, cell ortissue origins, host vs. pathogen, sample origins, subject origins, andthe like), different amplification products (e.g., amplificationproducts generated from different sets of primer pairs), or combinationsthereof. In some embodiments, a mixture of nucleic acids comprises aplurality amplification product species generated from different sets ofprimer pairs (e.g., 2 or more, 3 or more, 4 or more, 5 or more, 6 ormore, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 ormore, 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, 18 ormore, 19 or more, or 20 or more amplification product species). In someembodiments, a mixture of nucleic acids comprises single-strandednucleic acid and double-stranded nucleic acid. In some embodiment, amixture of nucleic acids comprises DNA and RNA. In some embodiment, amixture of nucleic acids comprises ribosomal RNA (rRNA) and messengerRNA (mRNA). Nucleic acid provided for processes described herein maycontain nucleic acid from one sample or from two or more samples (e.g.,from 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 ormore, 14 or more, 15 or more, 16 or more, 17 or more, 18 or more, 19 ormore, or 20 or more samples).

Nucleic acid may be derived from one or more plant sources by methodsknown in the art. Any suitable method can be used for isolating,extracting and/or purifying DNA from a plant sample, non-limitingexamples of which include methods of DNA preparation (e.g., described bySambrook and Russell, Molecular Cloning: A Laboratory Manual 3d ed.,2001), various commercially available reagents or kits, such as DNeasy®,RNeasy®, QIAprep®, QIAquick®, and QIAamp®, nucleic acidisolation/purification kits by Qiagen, Inc. (Germantown, Md.); DNAzol®,ChargeSwitch®, Purelink®, GeneCatcher® nucleic acidisolation/purification kits by Life Technologies, Inc. (Carlsbad,Calif.); NucleoMag®, NucleoSpin®, and NucleoBond® nucleic acidisolation/purification kits by Clontech Laboratories, Inc. (MountainView, Calif.), DNA/RNA extraction kits from Zymo Research (e.g.,ZYMOBIOMICS DNA Mini Kit, ZYMOBIOMICS DNA/RNA Miniprep Kit, ZYMOCLEANgel DNA recovery); the like or combinations thereof.

Nucleic acid may be provided for conducting methods described hereinwith or without processing of the sample(s) containing the nucleic acid.In some embodiments, nucleic acid is provided for conducting methodsdescribed herein after processing of the sample(s) containing thenucleic acid. For example, a nucleic acid can be extracted, isolated,purified, partially purified and/or amplified from the sample(s). Theterm “isolated” as used herein refers to nucleic acid removed from itsoriginal environment (e.g., the natural environment if it is naturallyoccurring, or a host cell if expressed exogenously), and thus is alteredby human intervention (e.g., “by the hand of man”) from its originalenvironment. The term “isolated nucleic acid” as used herein can referto a nucleic acid removed from a test subject (e.g., a plant). Anisolated nucleic acid can be provided with fewer non-nucleic acidcomponents (e.g., protein, lipid) than the amount of components presentin a source sample. A composition comprising isolated nucleic acid canbe about 50% to greater than 99% free of non-nucleic acid components. Acomposition comprising isolated nucleic acid can be about 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or greater than 99% free ofnon-nucleic acid components. The term “purified” as used herein canrefer to a nucleic acid provided that contains fewer non-nucleic acidcomponents (e.g., protein, lipid, carbohydrate) than the amount ofnon-nucleic acid components present prior to subjecting the nucleic acidto a purification procedure. A composition comprising purified nucleicacid may be about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater than 99% free ofother non-nucleic acid components. The term “purified” as used hereincan refer to a nucleic acid provided that contains fewer nucleic acidspecies than in the sample source from which the nucleic acid isderived. A composition comprising purified nucleic acid may be about90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater than 99%free of other nucleic acid species. In certain examples, pathogennucleic acid can be purified from a mixture comprising pathogen and hostnucleic acid. In certain examples, HpLVd or other plant pathogen genomicDNA can be purified from a mixture comprising HpLVd or other plantpathogen genomic DNA and Cannabis genomic DNA. In some embodiments,nucleic acid is provided for conducting methods described herein withoutprior processing of the sample(s) containing the nucleic acid. Forexample, nucleic acid may be analyzed directly from a sample withoutprior extraction, purification, partial purification, and/oramplification.

Nucleic acid also may be exposed to a process that modifies certainnucleotides in the nucleic acid before providing nucleic acid for amethod described herein. A process that selectively modifies nucleicacid based upon the methylation state of nucleotides therein can beapplied to nucleic acid, for example. In addition, conditions such ashigh temperature, ultraviolet radiation, x-radiation, can induce changesin the sequence of a nucleic acid molecule. In some embodiments, a plantis exposed to thermotherapy (heat treatment) prior to providing nucleicacid for a method described herein. Nucleic acid may be provided in anysuitable form useful for conducting an analysis (e.g., genotypeanalysis, sequence analysis).

Primers

Primers useful for detection, amplification, quantification, sequencingand/or analysis of nucleic acid are provided. The term “primer” as usedherein refers to a nucleic acid that includes a nucleotide sequencecapable of hybridizing or annealing to a target nucleic acid, at or near(e.g., adjacent to) a specific region of interest. Primers can allow forspecific determination of a target nucleic acid nucleotide sequence ordetection of the target nucleic acid (e.g., presence or absence of asequence), or feature thereof, for example. A primer typically is asynthetic sequence. The term “specific” or “specificity,” as usedherein, refers to the binding or hybridization of one molecule toanother molecule, such as a primer for a target polynucleotide. That is,“specific” or “specificity” refers to the recognition, contact, andformation of a stable complex between two molecules, as compared tosubstantially less recognition, contact, or complex formation of eitherof those two molecules with other molecules. As used herein, the terms“anneal” and “hybridize” refer to the formation of a stable complexbetween two molecules. The terms “primer,” “polynucleotide,” “oligo,” or“oligonucleotide” may be used interchangeably throughout the document,when referring to primers.

A primer nucleic acid can be designed and synthesized using suitableprocesses, and may be of any length suitable for hybridizing to anucleotide sequence of interest (e.g., where the nucleic acid is inliquid phase or bound to a solid support) and performing analysisprocesses described herein. Primers may be designed based upon a targetnucleotide sequence. A primer in some embodiments may be about 10 toabout 100 nucleotides, about 10 to about 70 nucleotides, about 10 toabout 50 nucleotides, about 15 to about 30 nucleotides, or about 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides inlength, or about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100nucleotides in length. A primer may be composed of naturally occurringand/or non-naturally occurring nucleotides (e.g., labeled nucleotides),or a mixture thereof. Primers suitable for use with embodimentsdescribed herein, may be synthesized and labeled using known techniques.Primers may be chemically synthesized according to the solid phasephosphoramidite triester method first described by Beaucage andCaruthers, Tetrahedron Letts., 22:1859-1862, 1981, using an automatedsynthesizer, as described in Needham-VanDevanter et al., Nucleic AcidsRes. 12:6159-6168, 1984. Purification of primers can be effected bynative acrylamide gel electrophoresis or by anion-exchangehigh-performance liquid chromatography (HPLC), for example, as describedin Pearson and Regnier, J. Chrom., 255:137-149, 1983.

In some embodiments, a primer provided herein (e.g., a further primerherein) comprises a polynucleotide where one or more nucleotidepositions contain a nonstandard nucleotide and/or a degeneratenucleotide. A nonstandard nucleotide may be, for example, a non-naturalbase, a modified base, or a universal base. A universal base is a basecapable of indiscriminately base pairing with each of the four standardnucleotide bases: A, C, G and T. Universal bases that may beincorporated into a primer herein include, but are not limited to,inosine, deoxyinosine, 2′-deoxyinosine (dl, dlnosine), nitroindole,5-nitroindole, and 3-nitropyrrole (e.g., 5′ nitroindole, deoxyinosine,deoxynebularine). A degenerate nucleotide typically refers to a mixtureof nucleotides at a given position and may be represented by a letterother than A, T, G or C. For example, a degenerate nucleotide may berepresented by R (A or G), Y (C or T), S (G or C), W (A or T), K (G orT), M (A or C), B (C or G or T), D (A or G or T), H (A or C or T), V (Aor C or G), or N (any base), for example. Such symbols for degeneratenucleotides are part of the International Union of Pure and AppliedChemistry (IUPAC) standard nomenclature for nucleotide base sequencenames and represent degenerate or nonstandard nucleotides that can bindmultiple nucleotides. For example, an “M” in a primer or probe wouldinclude a mixture of A and C at that position, and thus could bind toeither T or G in a complementary DNA strand. An “N” in a primer or probewould include a mixture of A, T, G and C at that position, and thuscould bind to any nucleotide at that position in the complementary DNAstrand.

All or a portion of a primer sequence may be complementary orsubstantially complementary to a target nucleic acid. As referred toherein, “substantially complementary” with respect to sequences refersto nucleotide sequences that will hybridize with each other. Thestringency of the hybridization conditions can be altered to toleratevarying amounts of sequence mismatch. Included are target and primersequences that are 55% or more, 56% or more, 57% or more, 58% or more,59% or more, 60% or more, 61% or more, 62% or more, 63% or more, 64% ormore, 65% or more, 66% or more, 67% or more, 68% or more, 69% or more,70% or more, 71% or more, 72% or more, 73% or more, 74% or more, 75% ormore, 76% or more, 77% or more, 78% or more, 79% or more, 80% or more,81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% ormore, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more,92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% ormore, 98% or more or 99% or more up to 100% complementary to each other.

Primers that are substantially complimentary to a target nucleic acidsequence are also substantially identical to the complement of thetarget nucleic acid sequence. That is, primers are substantiallyidentical to the anti-sense strand of the nucleic acid. As referred toherein, “substantially identical” with respect to sequences refers tonucleotide sequences that are 55% or more, 56% or more, 57% or more, 58%or more, 59% or more, 60% or more, 61% or more, 62% or more, 63% ormore, 64% or more, 65% or more, 66% or more, 67% or more, 68% or more,69% or more, 70% or more, 71% or more, 72% or more, 73% or more, 74% ormore, 75% or more, 76% or more, 77% or more, 78% or more, 79% or more,80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% ormore, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more,91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% ormore, 97% or more, 98% or more or 99% or more up to 100% identical toeach other. One test for determining whether two nucleotide sequencesare substantially identical is to determine the percent of identicalnucleotide sequences shared.

Primer sequences and length may affect hybridization to target nucleicacid sequences. Depending on the degree of mismatch between the primerand target nucleic acid, low, medium or high stringency conditions maybe used to effect primer/target annealing. As used herein, the term“stringent conditions” refers to conditions for hybridization andwashing. Methods for hybridization reaction temperature conditionoptimization are known and may be found in Current Protocols inMolecular Biology, John Wiley & Sons, N.Y., 6.3.1-6.3.6 (1989). Aqueousand non-aqueous methods are described in that reference and either canbe used. Non-limiting examples of stringent hybridization conditions arehybridization in 6× sodium chloride/sodium citrate (SSC) at about 45°C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 50° C.Another example of stringent hybridization conditions are hybridizationin 6× sodium chloride/sodium citrate (SSC) at about 45° C., followed byone or more washes in 0.2×SSC, 0.1% SDS at 55° C. A further example ofstringent hybridization conditions is hybridization in 6× sodiumchloride/sodium citrate (SSC) at about 45° C., followed by one or morewashes in 0.2×SSC, 0.1% SDS at 60° C. Often, stringent hybridizationconditions are hybridization in 6× sodium chloride/sodium citrate (SSC)at about 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at65° C. More often, stringency conditions are 0.5 M sodium phosphate, 7%SDS at 65° C., followed by one or more washes at 0.2×SSC, 1% SDS at 65°C. Stringent hybridization temperatures can also be altered (i.e.,lowered) with the addition of certain organic solvents, formamide forexample. Organic solvents, like formamide, reduce the thermal stabilityof double-stranded polynucleotides, so that hybridization can beperformed at lower temperatures, while still maintaining stringentconditions and extending the useful life of nucleic acids that may beheat labile. Features of primers described herein may also apply toprobes, such as, for example, the qPCR probes provided herein. Thereverse complement of each primer and probe described herein also iscontemplated herein.

As used herein, the phrase “hybridizing” or grammatical variationsthereof, refers to binding of a first nucleic acid molecule to a secondnucleic acid molecule under low, medium or high stringency conditions,or under nucleic acid synthesis conditions. Hybridizing can includeinstances where a first nucleic acid molecule binds to a second nucleicacid molecule, where the first and second nucleic acid molecules arecomplementary. As used herein, “specifically hybridizes” refers topreferential hybridization under nucleic acid synthesis conditions of aprimer, to a nucleic acid molecule having a sequence complementary tothe primer compared to hybridization to a nucleic acid molecule nothaving a complementary sequence. For example, specific hybridizationincludes the hybridization of a primer to a target nucleic acid sequencethat is complementary to the primer.

In some embodiments primers can include a nucleotide subsequence thatmay be complementary to a solid phase nucleic acid primer hybridizationsequence or substantially complementary to a solid phase nucleic acidprimer hybridization sequence (e.g., about 75%, 76%, 77%, 78%, 79%, 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or greater than 99% identical to the primerhybridization sequence complement when aligned). A primer may contain anucleotide subsequence not complementary to or not substantiallycomplementary to a solid phase nucleic acid primer hybridizationsequence (e.g., at the 3′ or 5′ end of the nucleotide subsequence in theprimer complementary to or substantially complementary to the solidphase primer hybridization sequence).

A primer, in certain embodiments, may contain a modification such as oneor more nonstandard nucleotides, non-natural nucleotides, universalbases, degenerate nucleotides, inosines, abasic sites, locked nucleicacids, minor groove binders, duplex stabilizers (e.g., acridine,spermidine), Tm modifiers or any modifier that changes the bindingproperties of the primers or probes. A primer, in certain embodiments,may contain a detectable molecule or entity (e.g., a fluorophore,radioisotope, colorimetric agent, particle, enzyme, and the like).

A primer also may refer to a polynucleotide sequence that hybridizes toa subsequence of a target nucleic acid or another primer and facilitatesthe detection of a primer, a target nucleic acid or both, as withmolecular beacons, for example. The term “molecular beacon” as usedherein refers to detectable molecule, where the detectable property ofthe molecule is detectable only under certain specific conditions,thereby enabling it to function as a specific and informative signal.Non-limiting examples of detectable properties are: optical properties,electrical properties, magnetic properties, chemical properties and timeor speed through an opening of known size.

Amplification

Nucleic acids may be amplified under amplification conditions. The term“amplify,” “amplification,” “amplification reaction,” “amplifying,”“amplified,” or “amplification conditions” as used herein refers tosubjecting a target nucleic acid (e.g., HpLVd, AMV, BCTV genomic DNA) ina sample to a process that linearly or exponentially generates ampliconnucleic acids having the same or substantially the same nucleotidesequence as the target nucleic acid (e.g., HpLVd, AMV, BCTV genomicDNA), or part (i.e., subsequence) thereof. In certain embodiments, theterm “amplified” or “amplification” or “amplification conditions” refersto a method that comprises a polymerase chain reaction (PCR). Nucleicacid may be amplified using a suitable amplification process. Nucleicacid amplification typically involves enzymatic synthesis of nucleicacid amplicons (copies), which contain a sequence complementary to anucleotide sequence being amplified.

In some embodiments a limited amplification reaction, also known aspre-amplification, can be performed. Pre-amplification is a method inwhich a limited amount of amplification occurs due to a small number ofcycles, for example 10 cycles, being performed. Pre-amplification canallow some amplification, but stops amplification prior to theexponential phase, and typically produces about 500 copies of thedesired nucleotide sequence(s). Use of pre-amplification may also limitinaccuracies associated with depleted reactants in standard PCRreactions, for example, and also may reduce amplification biases due tonucleotide sequence or species abundance of the target. In someembodiments, a one-time primer extension may be used may be performed asa prelude to linear or exponential amplification.

Any suitable amplification technique can be utilized. Amplification ofmethods include, but are not limited to, polymerase chain reaction(PCR); ligation amplification (or ligase chain reaction (LCR));amplification methods based on the use of Q-beta replicase ortemplate-dependent polymerase (e.g., U.S. Patent Publication NumberUS20050287592); helicase-dependent isothermal amplification (Vincent etal., “Helicase-dependent isothermal DNA amplification”. EMBO reports 5(8): 795-800 (2004)); strand displacement amplification (SDA);thermophilic SDA nucleic acid sequence based amplification (3SR orNASBA), and transcription-associated amplification (TAA). Non-limitingexamples of PCR amplification methods include standard PCR, AFLP-PCR,allele-specific PCR, Alu-PCR, asymmetric PCR, colony PCR, hot start PCR,inverse PCR (IPCR), in situ PCR (ISH), intersequence-specific PCR(ISSR-PCR), long PCR, multiplex PCR, nested PCR, quantitative PCR(qPCR), reverse transcriptase PCR (RT-PCR), reverse transcriptasequantitative PCR (RT-qPCR), TAQMAN qPCR, real time PCR, single cell PCR,solid phase PCR, combinations thereof, and the like. Reagents andhardware for conducting PCR are commercially available.

A generalized description of an amplification process is as follows.Primers and target nucleic acid are contacted, and complementarysequences hybridize to one another, for example. Primers can hybridizeto a target nucleic acid, at or near (e.g., adjacent to, abutting, andthe like) a sequence of interest. A reaction mixture, containingcomponents necessary for enzymatic functionality, is added to theprimer-target nucleic acid hybrid, and amplification can occur undersuitable conditions. Components of an amplification reaction mayinclude, but are not limited to, e.g., primers (e.g., individualprimers, primer pairs, a plurality of primer pairs, and the like) apolynucleotide template (e.g., target nucleic acid), polymerase,nucleotides, dNTPs and the like. In some embodiments, non-naturallyoccurring nucleotides or nucleotide analogs, such as analogs containinga detectable label (e.g., fluorescent or colorimetric label), may beused for example. Any suitable polymerase may be selected which mayinclude polymerases for thermocycle amplification (e.g., Taq DNAPolymerase; Q-Bio™ Taq DNA Polymerase (recombinant truncated form of TaqDNA Polymerase lacking 5′-3′exo activity); SurePrime™ Polymerase(chemically modified Taq DNA polymerase for “hot start” PCR); Arrow™ TaqDNA Polymerase (high sensitivity and long template amplification)) andpolymerases for thermostable amplification (e.g., RNA polymerase fortranscription-mediated amplification (TMA) described at World Wide WebURL “gen-probe.com/pdfs/tma_whiteppr.pdf”). Other enzyme components canbe added, such as reverse transcriptase for transcription mediatedamplification (TMA) reactions, for example.

PCR conditions can be dependent upon primer sequences, target abundance,and the desired amount of amplification, and therefore, any suitable PCRprotocol may be selected. PCR is typically carried out as an automatedprocess with a thermostable enzyme. In this process, the temperature ofthe reaction mixture is cycled through a denaturing step, aprimer-annealing step, and an extension reaction step automatically.Some PCR protocols also include an activation step and a final extensionstep. Machines specifically adapted for this purpose are commerciallyavailable. A non-limiting example of a PCR protocol that may be suitablefor embodiments described herein is as follows: treating the sample at95° C. for 2 minutes; repeating 40 cycles of 95° C. for 15 seconds and60° C. for 30 seconds. Additional examples of suitable PCR protocols areprovided in Examples 1 and 2. A completed PCR reaction can optionally bekept at 4° C. until further action is desired. Multiple cyclesfrequently are performed using a commercially available thermal cycler.Suitable isothermal amplification processes also may be applied, incertain embodiments.

In some embodiments, an amplification product may include naturallyoccurring nucleotides, non-naturally occurring nucleotides, nucleotideanalogs and the like and combinations of the foregoing. An amplificationproduct often has a nucleotide sequence that is identical to orsubstantially identical to a sample nucleic acid nucleotide sequence orcomplement thereof. A “substantially identical” nucleotide sequence inan amplification product will generally have a high degree of sequenceidentity to the nucleotide sequence species being amplified orcomplement thereof (e.g., about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or greater than 99% sequence identity), and variationssometimes are a result of infidelity of the polymerase used forextension and/or amplification, or additional nucleotide sequence(s)added to the primers used for amplification.

In some embodiments where a target nucleic acid is RNA, prior to theamplification step, a DNA copy (cDNA) of the RNA transcript of interestmay be synthesized. A cDNA can be synthesized by reverse transcription,which can be carried out as a separate step, or in a homogeneous reversetranscription-polymerase chain reaction (RT-PCR), a modification of thepolymerase chain reaction for amplifying RNA.

Amplification also can be accomplished using digital PCR, in certainembodiments. Digital PCR takes advantage of nucleic acid (DNA, cDNA orRNA) amplification on a single molecule level, and offers a highlysensitive method for quantifying low copy number nucleic acid. Systemsfor digital amplification and analysis of nucleic acids are available(e.g., Fluidigm® Corporation).

Amplification reactions may be performed as individual amplificationreactions, where one primer pair is used for each reaction and thepresence or absence of one amplification product is detected. In someembodiments, multiple individual amplification reactions may beperformed (i.e., carried out in separate containers) using a differentset of primers for each reaction, and the presence or absence of anamplification product is detected for each individual reaction. In someembodiments, amplification reactions are performed as multiplexamplification reactions (i.e., a plurality of amplification reactionsperformed in a single container), where a plurality of primer pairs isused for the multiplex reaction, and the presence or absence of morethan one amplification product is detected. Both individualamplification reactions and multiplex amplification reactions arecontemplated for the primers provided herein.

In some embodiments, when the plant pathogen is HpLVd, a method hereincomprises generating nucleic acid amplification products from a plantsample. Such method may comprise a) contacting nucleic acid of a plantsample with a first set of polynucleotide primers under amplificationconditions, thereby generating a first set of amplification products,where i) the majority or all of the primers in the first set ofpolynucleotide primers hybridize to subsequences of SEQ ID NO:1 ifpresent in the nucleic acid of the plant sample under the amplificationconditions, ii) the subsequences of SEQ ID NO:1 to which the majority orall of the primers in the first set of polynucleotide primers hybridizeunder the amplification conditions contain no variant nucleotideposition, and iii) each subsequence of SEQ ID NO:1 between thesubsequences to which the primers in the first set of polynucleotideprimers hybridize contain one or more variant nucleotide positions; andb) contacting the nucleic acid of the plant sample with a second set ofpolynucleotide primers under the amplification conditions, therebygenerating a second set of amplification products, where i) the majorityor all of the primers in the second set of polynucleotide primershybridize to subsequences of SEQ ID NO:1 if present in the nucleic acidof the plant sample under the amplification conditions, and ii) thesubsequences of SEQ ID NO:1 to which the majority or all of the primersin the second set of polynucleotide primers hybridize under theamplification conditions contain one or more variant nucleotidepositions. In some embodiments, a method herein comprises analyzing thefirst and second sets of amplification products.

Quantitative PCR

In some embodiments, an amplification method comprises a quantifiableamplification method. For example, levels of HpLVd, AMV, BCTV or otherplant pathogen may be measured using a quantitative PCR (qPCR) approach(e.g., on cDNA generated from RNA from a plant sample), or a reversetranscriptase quantitative PCR (RT-qPCR) approach (e.g., on RNA from aplant sample). Quantitative PCR (qPCR), which also may be referred to areal-time PCR, monitors the amplification of a targeted nucleic acidmolecule during a PCR reaction (i.e., in real time). This method may beused quantitatively (quantitative real-time PCR) and semi-quantitatively(i.e., above/below a certain amount of nucleic acid molecules;semi-quantitative real-time PCR).

Methods for qPCR include use of non-specific fluorescent dyes thatintercalate with double-stranded DNA, and sequence-specific DNA probeslabelled with a fluorescent reporter, which generally allows detectionafter hybridization of the probe with its complementary sequence.Quantitative PCR methods typically are performed in a thermal cyclerwith the capacity to illuminate each sample with a beam of light of atleast one specified wavelength and detect the fluorescence emitted by anexcited fluorophore.

For non-specific detection, a DNA-binding dye binds to alldouble-stranded (ds) DNA during PCR. An increase in DNA product duringPCR therefore leads to an increase in fluorescence intensity measured ateach cycle. For qPCR using dsDNA dyes, the reaction typically isprepared like a basic PCR reaction, with the addition of fluorescentdsDNA dye. Then the reaction is run in a real-time PCR instrument, andafter each cycle, the intensity of fluorescence is measured with adetector (the dye only fluoresces when bound to the dsDNA (i.e., the PCRproduct)). In certain applications, multiple target sequences may bemonitored in a tube by using different types of dyes.

For specific detection, fluorescent reporter probes detect only the DNAcontaining the sequence complementary to the probe. Accordingly, use ofthe reporter probe increases specificity, and enables performing thetechnique even in the presence of other dsDNA. Using different types oflabels, fluorescent probes may be used in multiplex assays formonitoring several target sequences in the same tube. This methodtypically uses a DNA-based probe with a fluorescent reporter at one endand a quencher of fluorescence at the opposite end of the probe. Theclose proximity of the reporter to the quencher prevents detection ofits fluorescence. During PCR, the probe is broken down by the 5′ to 3′exonuclease activity of the polymerase, which breaks thereporter-quencher proximity and thus allows unquenched emission offluorescence, which can be detected after excitation with a laser. Anincrease in the product targeted by the reporter probe at each PCR cycletherefore causes a proportional increase in fluorescence due to thebreakdown of the probe and release of the reporter.

In some embodiments, a method herein comprises contacting nucleic acidof a plant sample with one or more primer pairs and one or morequantitative PCR probes. Polynucleotide primers and polynucleotideprobes can be designed and or used as provided herein, e.g., todetermine the presence, absence and/or amount of a pathogen in a plant.

For example, when the pathogen is HpLVd, certain primers provided herein(e.g., primers provided in Table 1) may be used in combination withcertain qPCR probes (e.g., probes provided in Table 5). Examples ofspecific combinations of primers and probes that can identify HpLVd in aplant sample are provided in Table 4. These combinations may be used ona cDNA template or an RNA template that is extracted from the plant. Insome embodiments, one or more quantitative PCR probes are chosen fromone or more of TCGTGCGCGGCGACCT (SEQ ID NO:16), CGGAGATCGAGCGCCAGTT (SEQID NO:17), TGCGCGGCGACCTGAAGT (SEQ ID NO:18), AGGCGGAGATCGAGCGCCA (SEQID NO:19), and TCCTGCGTGGAACGGCTCC (SEQ ID NO:20). The reversecomplement of each of the probes also is contemplated herein.

In some embodiments, a quantitative PCR probe (e.g., a probe set forthas SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, and/or SEQ IDNO:20) comprises a polynucleotide where one or more nucleotide positionscontain a nonstandard nucleotide and/or a degenerate nucleotide.Nonstandard nucleotides and degenerate nucleotide are described above.In some embodiments, a quantitative PCR probe (e.g., a probe set forthas SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, and/or SEQ IDNO:20) comprises a polynucleotide where two or more nucleotide positionscontain a nonstandard nucleotide and/or a degenerate nucleotide. In someembodiments, a quantitative PCR probe (e.g., a probe set forth as SEQ IDNO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, and/or SEQ ID NO:20)comprises a polynucleotide where three or more nucleotide positionscontain a nonstandard nucleotide and/or a degenerate nucleotide. In someembodiments, a quantitative PCR probe (e.g., a probe set forth as SEQ IDNO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, and/or SEQ ID NO:20)comprises a polynucleotide where four or more nucleotide positionscontain a nonstandard nucleotide and/or a degenerate nucleotide. In someembodiments, a quantitative PCR probe (e.g., a probe set forth as SEQ IDNO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, and/or SEQ ID NO:20)comprises a polynucleotide where five or more nucleotide positionscontain a nonstandard nucleotide and/or a degenerate nucleotide.

Loop Mediated Isothermal Amplification (LAMP)

In some embodiments, an amplification method comprises loop mediatedisothermal amplification (LAMP). Loop-mediated isothermal amplification(LAMP) is a single-tube technique useful for nucleic acid amplification.Reverse transcription loop-mediated isothermal amplification (RT-LAMP)combines LAMP with a reverse transcription step for the detection ofRNA. LAMP is typically performed under isothermal conditions. Incontrast to a polymerase chain reaction (PCR) technology, which istypically performed using a series of alternating temperature cycles,isothermal amplification is performed at a constant temperature, anddoes not require a thermal cycler.

In LAMP, a target sequence is amplified at a constant temperature (e.g.,between about 60° C. to about 65° C.) using a plurality of primer pairs(e.g., two primer pairs, three primer pairs) and a polymerase (e.g., apolymerase with high strand displacement activity). In certainapplications, four different primers may be used to amplify six distinctregions on a target sequence, for example, which may increasespecificity. An additional pair of loop primers can further acceleratethe reaction.

The amplification product can be detected via photometry (i.e.,measuring the turbidity caused by magnesium pyrophosphate precipitate insolution as a byproduct of amplification). This generally allows forvisualization by the naked eye or by photometric detection approaches(e.g., for small volumes). In certain applications, the reaction can befollowed in real-time either by measuring turbidity or by fluorescenceusing intercalating dyes (e.g., SYTO 9, SYBR green). Certain dyes may beused to create a visible color change that can be seen with the nakedeye without the need for specialized equipment. Dye moleculesintercalate or directly label the DNA, and in turn can be correlatedwith the number of copies initially present. Accordingly, certainvariations of LAMP may be quantitative. Detection of LAMP amplificationproducts also may be achieved using manganese loaded calcein, whichstarts fluorescing upon complexation of manganese by pyrophosphateduring in vitro DNA synthesis. Another method for visual detection ofLAMP amplification products by the naked eye is based on the ability ofthe products to hybridize with complementary gold-bound single-strandedDNA, which prevents a red to purple-blue color change that wouldotherwise occur during salt-induced aggregation of the gold particles.

A number of LAMP visualization technologies are known to those of skillin the art (see, e.g., Fischbach et al., Biotechniques, 58(4):189-194(2015), the contents of which are incorporated in their entirety byreference herein). Examples of such visualization reagents, summarizedin the Table below from Fischbach et al., include magnesiumpyrophosphate, hydroxynaphthol blue (HNB), calcein, SYBR Green I,EvaGreen and the nucleic acid-specific dye, berberine, which emits afluorescent signal under UV light after a positive LAMP reaction.

indicates data missing or illegible when filed

In some embodiments, a method herein comprises contacting nucleic acidof a plant sample with a set of loop mediated isothermal amplification(LAMP) primers. For example, when the pathogen is HpLVd, a method hereinmay comprise contacting nucleic acid of a plant sample with a set ofloop mediated isothermal amplification (LAMP) primers chosen from theprimer sets provided in Tables 6-9 herein. In some embodiments, a LAMPprimer set comprises the polynucleotides of SEQ ID NO:21 to SEQ IDNO:29. In some embodiments, a LAMP primer set comprises thepolynucleotides of SEQ ID NO:30 to SEQ ID NO:38. In some embodiments, aLAMP primer set comprises the polynucleotides of a primer set comprisingthe polynucleotides of SEQ ID NO:39 to SEQ ID NO:47. In someembodiments, a LAMP primer set comprises the polynucleotides of SEQ IDNO:48 to SEQ ID NO:56.

Detection of Amplification Products

Amplification products generated by a method herein may be detected by asuitable detection process. Non-limiting examples of methods ofdetection include electrophoresis, nucleic acid sequencing, massspectrometry, mass detection of mass modified amplicons (e.g.,matrix-assisted laser desorption ionization (MALDI) mass spectrometryand electrospray (ES) mass spectrometry), a primer extension method(e.g., iPLEX™; Sequenom, Inc.), Molecular Inversion Probe (MIP)technology from Affymetrix, restriction fragment length polymorphism(RFLP analysis), allele specific oligonucleotide (ASO) analysis,methylation-specific PCR (MSPCR), pyrosequencing analysis, acycloprimeanalysis, Reverse dot blot, GeneChip microarrays, Dynamicallele-specific hybridization (DASH), Peptide nucleic acid (PNA) andlocked nucleic acids (LNA) probes, TaqMan, Molecular Beacons,Intercalating dye, FRET primers, AlphaScreen, SNPstream, genetic bitanalysis (GBA), Multiplex minisequencing, SNaPshot, GOOD assay,Microarray miniseq, arrayed primer extension (APEX), Microarray primerextension, Tag arrays, coded microspheres, template-directedincorporation (TDI), fluorescence polarization, colorimetricoligonucleotide ligation assay (OLA), sequence-coded OLA, microarrayligation, ligase chain reaction, padlock probes, invader assay,hybridization using at least one probe, hybridization using at least onefluorescently labeled probe, cloning and sequencing, the use ofhybridization probes and quantitative real time polymerase chainreaction (QRT-PCR), digital PCR, nanopore sequencing, chips, MYBAIT(Arbor Biosciences), SNPCHIP, various microarray platforms, andcombinations thereof.

In some embodiments, amplification products are detected usingelectrophoresis. Any suitable electrophoresis method, whereby amplifiednucleic acids are separated by size, may be used in conjunction with themethods provided herein, which include, but are not limited to, standardelectrophoretic techniques and specialized electrophoretic techniques,such as, for example capillary electrophoresis (e.g., Capillary ZoneElectrophoresis (CZE), also known as free-solution CE (FSCE), CapillaryIsoelectric Focusing (CIEF), Isotachophoresis (ITP), ElectrokineticChromatography (EKC), Micellar Electrokinetic Capillary Chromatography(MECC OR MEKC), Micro Emulsion Electrokinetic Chromatography (MEEKC),Non-Aqueous Capillary Electrophoresis (NACE), and CapillaryElectrochromatography (CEC)). A non-limiting standard electrophoresisexample is presented as follows. After running an amplified nucleic acidsample in an agarose or polyacrylamide gel, the gel may be labeled(e.g., stained) with ethidium bromide (see, Sambrook and Russell,Molecular Cloning: A Laboratory Manual 3d ed., 2001). The presence of aband of the same size as the standard control is an indication of thepresence of a target nucleic acid sequence, the amount of which may thenbe compared to the control based on the intensity of the band, thusdetecting and quantifying the target sequence of interest. In someembodiments, where a plurality of primer pairs is used in anamplification reaction, multiple amplification products of varying sizemay be detected using electrophoresis.

High Resolution Melting (HRM)

In some embodiments, nucleic acid is analyzed using a high resolutionmelting (HRM) endpoint assay. In some embodiments, an analysis comprisesperforming a high resolution melting (HRM) endpoint assay onamplification products (e.g., amplification products generated usingprimers provided herein). In some embodiments, an analysis comprisesperforming a high resolution melting (HRM) endpoint assay on nucleicacid in a mixture (e.g., a mixture of amplification products generatedusing a plurality of primer pairs).

High resolution melt or high resolution melting (HRM) analysis is atechnique useful for the detection of mutations, polymorphisms, andepigenetic differences in double-stranded DNA. Typically, amplification(e.g., a polymerase chain reaction (PCR)) is performed prior to HRManalysis to amplify a DNA region in which a mutation of interest islocated. The HRM process involves a precise warming of the amplificationproduct from around 50° C. up to around 95° C. At some point during thisprocess, the melting temperature of the amplicon is reached and the twostrands of DNA separate (i.e., melt apart).

The separation of strands may be monitored in real-time (e.g., using afluorescent dye). Dyes that may be used for HRM include intercalatingdyes, which specifically bind to double-stranded DNA and emitfluorescence when bound to DNA. At the start of an HRM analysis there isa high level of fluorescence in the sample because of the billions ofcopies of the amplicon. However, as the sample is heated up and the twostrands of the DNA melt apart, presence of double stranded DNAdecreases, and thus the fluorescence is reduced. In certainconfigurations, an HRM machine has a camera that monitors this processby measuring the fluorescence. The machine can plot the data (e.g., as agraph sometimes referred to as a melt curve), showing the level offluorescence vs. temperature.

The melting temperature of an amplification product at which the two DNAstrands come apart is a predictable parameter, and typically isdependent on the DNA sequence of the amplicon. When comparing twosamples from two different plants infected with HpLVd or other plantpathogen, for example, amplification products from both samples shouldhave the same shaped melt curve. However, if one plant is infected withan HpLVd or other pathogen carrying a mutation in the amplified region,this will alter the temperature at which the DNA strands melt apart.Accordingly, the two melt curves will be different. The difference maybe subtle, but because HRM machines typically are capable of monitoringthe HRM process in high resolution, it is generally possible toaccurately document these changes and therefore identify if a mutationis present or not.

In some embodiments, an analysis comprises detecting one or more geneticvariations (e.g., single nucleotide substitutions) in a hops latentviroid or other pathogen according to results obtained from a highresolution melting (HRM) endpoint assay. In some embodiments, ananalysis comprises detecting two or more genetic variations (e.g.,single nucleotide substitutions) in a hops latent viroid or otherpathogen according to results obtained from a high resolution melting(HRM) endpoint assay. In some embodiments, an analysis comprisesdetecting three or more genetic variations (e.g., single nucleotidesubstitutions) in a hops latent viroid or other pathogen according toresults obtained from a high resolution melting (HRM) endpoint assay. Insome embodiments, an analysis comprises detecting four or more geneticvariations (e.g., single nucleotide substitutions) in a hops latentviroid or other pathogen according to results obtained from a highresolution melting (HRM) endpoint assay. In some embodiments, ananalysis comprises detecting five or more genetic variations (e.g.,single nucleotide substitutions) in a hops latent viroid or otherpathogen according to results obtained from a high resolution melting(HRM) endpoint assay. In some embodiments, an analysis comprisesdetecting six or more genetic variations (e.g., single nucleotidesubstitutions) in a hops latent viroid or other pathogen according toresults obtained from a high resolution melting (HRM) endpoint assay. Insome embodiments, an analysis comprises detecting seven or more geneticvariations (e.g., single nucleotide substitutions) in a hops latentviroid or other pathogen according to results obtained from a highresolution melting (HRM) endpoint assay. In some embodiments, ananalysis comprises detecting eight or more genetic variations (e.g.,single nucleotide substitutions) in a hops latent viroid or otherpathogen according to results obtained from a high resolution melting(HRM) endpoint assay. In some embodiments, an analysis comprisesdetecting nine or more genetic variations (e.g., single nucleotidesubstitutions) in a hops latent viroid or other pathogen according toresults obtained from a high resolution melting (HRM) endpoint assay. Insome embodiments, an analysis comprises detecting ten or more geneticvariations (e.g., single nucleotide substitutions) in a hops latentviroid or other pathogen according to results obtained from a highresolution melting (HRM) endpoint assay.

Nucleic Acid Sequencing

In some embodiments, nucleic acid is sequenced. In some embodiments,amplified subsequences of HpLVd, AMV, BCTV or other plant pathogens(“amplification products”) are sequenced by a sequencing process. Insome embodiments, the sequencing process generates sequence reads (orsequencing reads). In some embodiments, a method herein comprisesdetermining the sequence of an HpLVd, AMV, BCTV or other plant pathogensubsequence based on the sequence reads. In some embodiments, a methodherein comprises determining the sequence of an HpLVd, AMV, BCTV orother plant pathogen genome based on the sequence reads. In someembodiments, a method herein comprises determining one or more HpLVd,AMV, BCTV or other genotypes based on the sequence reads.

Nucleic acid may be sequenced using any suitable sequencing platform,non-limiting examples of which include Maxim & Gilbert,chain-termination methods, sequencing by synthesis, sequencing byligation, sequencing by mass spectrometry, microscopy-based techniques,the like or combinations thereof. In some embodiments, afirst-generation technology, such as, for example, Sanger sequencingmethods including automated Sanger sequencing methods, includingmicrofluidic Sanger sequencing, can be used in a method provided herein.In some embodiments, sequencing technologies that include the use ofnucleic acid imaging technologies (e.g., transmission electronmicroscopy (TEM) and atomic force microscopy (AFM)), can be used. Insome embodiments, a high-throughput sequencing method is used.High-throughput sequencing methods generally involve clonally amplifiedDNA templates or single DNA molecules that are sequenced in a massivelyparallel fashion, sometimes within a flow cell. Next generation (e.g.,2nd and 3rd generation) sequencing techniques capable of sequencing DNAin a massively parallel fashion can be used for methods described hereinand are collectively referred to herein as “massively parallelsequencing” (MPS). In some embodiments, MPS sequencing methods utilize atargeted approach, where specific chromosomes, genes or regions ofinterest are sequenced. For example, a targeted approach may includetargeting specific regions of an HpLVd, AMV, BCTV or other plantpathogen genome for sequencing. In certain embodiments, a non-targetedapproach is used where most or all nucleic acids in a sample aresequenced, amplified and/or captured randomly.

Non-limiting examples of sequencing platforms include a sequencingplatform provided by Illumina® (e.g., HiSeq™ HiSeq™ 2000, MiSeq™ GenomeAnalyzer™, and Genome Analyzer™ 11 sequencing systems); Oxford Nanopore™Technologies (e.g., MinION sequencing system), Ion Torrent™ (e.g., IonPGM™ and/or Ion Proton™ sequencing systems); Pacific Biosciences (e.g.,PACBIO RS 11 sequencing system); Life Technologies™ (e.g., SOLiDsequencing system); Roche (e.g., 454 GS FLX+ and/or GS Junior sequencingsystems); Helicos True Single Molecule Sequencing; Ionsemiconductor-based sequencing (e.g., as developed by LifeTechnologies), WildFire, 5500, 5500xl W and/or 5500xl W Genetic Analyzerbased technologies (e.g., as developed and sold by Life Technologies,U.S. Patent Application Publication No. 2013/0012399); Polonysequencing, Pyrosequencing, Massively Parallel Signature Sequencing(MPSS), RNA polymerase (RNAP) sequencing, LaserGen systems and methods,Nanopore-based platforms, chemical-sensitive field effect transistor(CHEMFET) array, electron microscopy-based sequencing (e.g., asdeveloped by ZS Genetics, Halcyon Molecular), nanoball sequencing; orany other suitable sequencing platform. Other sequencing methods thatmay be used to conduct methods herein include digital PCR, sequencing byhybridization, nanopore sequencing, chromosome-specific sequencing(e.g., using DANSR (digital analysis of selected regions) technology),MYBAIT (Arbor Biosciences), SNPCHIP, and microarray platforms.

In some embodiments, the sequencing process is a highly multiplexedsequencing process. In certain instances, a full or substantially fullsequence is obtained and sometimes a partial sequence is obtained.Nucleic acid sequencing generally produces a collection of sequencereads. As used herein, “reads” (e.g., “a read,” “a sequence read”) areshort sequences of nucleotides produced by any sequencing processdescribed herein or known in the art. Reads can be generated from oneend of nucleic acid fragments (single-end reads), and sometimes aregenerated from both ends of nucleic acid fragments (e.g., paired-endreads, double-end reads). In some embodiments, a sequencing processgenerates short sequencing reads or “short reads.” In some embodiments,the nominal, average, mean or absolute length of short reads sometimesis about 10 continuous nucleotides to about 250 or more contiguousnucleotides. In some embodiments, the nominal, average, mean or absolutelength of short reads sometimes is about 50 continuous nucleotides toabout 150 or more contiguous nucleotides.

The length of a sequence read is often associated with the particularsequencing technology utilized. High-throughput methods, for example,provide sequence reads that can vary in size from tens to hundreds ofbase pairs (bp). Nanopore sequencing, for example, can provide sequencereads that can vary in size from tens to hundreds to thousands of basepairs. In some embodiments, sequence reads are of a mean, median,average or absolute length of about 15 bp to about 900 bp long. Incertain embodiments sequence reads are of a mean, median, average orabsolute length of about 1000 bp or more. In some embodiments, sequencereads are of a mean, median, average or absolute length of about 100 bpto about 200 bp.

Reads generally are representations of nucleotide sequences in aphysical nucleic acid. For example, in a read containing an ATGCdepiction of a sequence, “A” represents an adenine nucleotide, “T”represents a thymine nucleotide, “G” represents a guanine nucleotide and“C” represents a cytosine nucleotide, in a physical nucleic acid.

In certain embodiments, “obtaining” nucleic acid sequence reads of asample from a plant and/or “obtaining” nucleic acid sequence reads fromone or more amplification products can involve directly sequencingnucleic acid to obtain the sequence information. In some embodiments,“obtaining” can involve receiving sequence information obtained directlyfrom a nucleic acid by another.

In some embodiments, some or all nucleic acids in a sample are enrichedand/or amplified (e.g., non-specifically, or specifically usingamplification primers described herein) prior to or during sequencing.In certain embodiments, specific nucleic acid species or subsets in asample are enriched and/or amplified prior to or during sequencing. Insome embodiments, nucleic acid from a pathogen may be enriched and/oramplified prior to or during sequencing, while nucleic acid from a hostplant is not enriched and/or amplified prior to or during sequencing.For example, nucleic acid from the HpLVd, AMV, BCTV or other plantpathogen genome may be enriched and/or amplified prior to or duringsequencing, while nucleic acid from the cannabis genome is not enrichedand/or amplified prior to or during sequencing. In some embodiments,nucleic acids in a sample are not enriched and/or amplified prior to orduring sequencing.

In some embodiments, one nucleic acid sample from one plant issequenced. In certain embodiments, nucleic acids from each of two ormore samples are sequenced, where samples are from one plant or fromdifferent plants. In certain embodiments, nucleic acid samples from twoor more biological samples are pooled, where each biological sample isfrom one plant or two or more plants, and the pool is sequenced. In thelatter embodiments, a nucleic acid sample from each biological sampleoften is identified by one or more unique identifiers.

A sequencing method may utilize identifiers that allow multiplexing ofsequence reactions in a sequencing process. The greater the number ofunique identifiers, the greater the number of samples and/or chromosomesfor detection, for example, that can be multiplexed in a sequencingprocess. A sequencing process can be performed using any suitable numberof unique identifiers (e.g., 4, 8, 12, 24, 48, 96, or more).

A sequencing process sometimes makes use of a solid phase, and sometimesthe solid phase comprises a flow cell on which nucleic acid from alibrary can be attached and reagents can be flowed and contacted withthe attached nucleic acid. A flow cell sometimes includes flow celllanes, and use of identifiers can facilitate analyzing a number ofsamples in each lane. A flow cell often is a solid support that can beconfigured to retain and/or allow the orderly passage of reagentsolutions over bound analytes. Flow cells frequently are planar inshape, optically transparent, generally in the millimeter orsub-millimeter scale, and often have channels or lanes in which theanalyte/reagent interaction occurs. In some embodiments, the number ofsamples analyzed in a given flow cell lane is dependent on the number ofunique identifiers utilized during library preparation and/or probedesign. Multiplexing using 12 identifiers, for example, allowssimultaneous analysis of 96 samples (e.g., equal to the number of wellsin a 96 well microwell plate) in an 8-lane flow cell. Similarly,multiplexing using 48 identifiers, for example, allows simultaneousanalysis of 384 samples (e.g., equal to the number of wells in a 384well microwell plate) in an 8-lane flow cell. Non-limiting examples ofcommercially available multiplex sequencing kits include Illumina'smultiplexing sample preparation oligonucleotide kit and multiplexingsequencing primers and PhiX control kit (e.g., Illumina's catalognumbers PE-400-1001 and PE-400-1002, respectively).

In some embodiments a targeted enrichment, amplification and/orsequencing approach is used. A targeted approach often isolates, selectsand/or enriches a subset of nucleic acids in a sample for furtherprocessing by use of sequence-specific oligonucleotides. In someembodiments, a library of sequence-specific oligonucleotides areutilized to target (e.g., hybridize to) one or more sets of nucleicacids in a sample. Sequence-specific oligonucleotides and/or primers areoften selective for particular sequences (e.g., unique nucleic acidsequences) present in one or more chromosomes, genes, exons, introns,and/or regulatory regions of interest. For example, primers specific forthe HpLVd, AMV, BCTV or other plant pathogen genome may be used for atargeted enrichment, amplification and/or sequencing approach. Anysuitable method or combination of methods can be used for enrichment,amplification and/or sequencing of one or more subsets of targetednucleic acids. In some embodiments targeted sequences are isolatedand/or enriched by capture to a solid phase (e.g., a flow cell, a bead)using one or more sequence-specific anchors. In some embodimentstargeted sequences are enriched and/or amplified by a polymerase-basedmethod (e.g., a PCR-based method, by any suitable polymerase-basedextension) using sequence-specific primers and/or primer sets (e.g.,primers provided herein). Sequence specific anchors often can be used assequence-specific primers.

In some embodiments, nucleic acid is sequenced and the sequencingproduct (e.g., a collection of sequence reads) is processed prior to, orin conjunction with, an analysis of the sequenced nucleic acid. Forexample, sequence reads may be processed according to one or more of thefollowing: aligning, mapping, filtering, counting, normalizing,weighting, generating a profile, and the like, and combinations thereof.Certain processing steps may be performed in any order and certainprocessing steps may be repeated.

Classifications and Uses Thereof

Methods described herein can provide an outcome indicative of one ormore characteristics of a sample or pathogen described above. In someembodiments, methods described herein can provide an outcome indicativeof one or more characteristics of a plant. In some embodiments, methodsdescribed herein can provide an outcome indicative of one or morecharacteristics of a cannabis plant. In some embodiments, methodsdescribed herein can provide an outcome indicative of one or morecharacteristics of a pathogen. In some embodiments, methods describedherein can provide an outcome indicative of one or more characteristicsof an HpLVd variant, an AMV variant, a BCTV variant, or other plantpathogen variant. Methods described herein sometimes provide an outcomeindicative of a phenotype and/or presence or absence of a pathogen for atest sample (e.g., providing an outcome determinative of the presence orabsence of a pathogen and/or phenotype, and/or an amount of a pathogen).For example, methods described herein sometimes provide an outcomeindicative of a phenotype (e.g., a phenotype expressed by the plant andassociated with an HpLVd, AMV, BCTV or other plant pathogen infection)and/or presence or absence of an HpLVd, AMV, BCTV or other plantpathogen infection for a plant sample (e.g., providing an outcomedeterminative of the presence or absence of an HpLVd, AMV, BCTV or otherplant pathogen infection and/or phenotype associated with an HpLVd, AMV,BCTV or other plant pathogen infection). An outcome often is part of aclassification process, and a classification (e.g., classification ofone or more characteristics of a sample; classification of one or morecharacteristics of a pathogen (e.g., HpLVd, AMV, BCTV or other plantpathogen); classification of one or more phenotypes associated with apathogen (e.g., HpLVd, AMV, BCTV or other plant pathogen);classification of one or more phenotypes associated with a particularvariant of a pathogen (e.g., an HpLVd, AMV, BCTV or other plant pathogenvariant); presence or absence of a genotype, phenotype, geneticvariation, and/or infection (e.g., an HpLVd, AMV, BCTV or other plantpathogen infection) for a test sample (e.g., a Cannabis plant sample);presence or absence of a genotype, phenotype, genetic variation, and/orgenetic variation signature for a pathogen (e.g., HpLVd, AMV, BCTV orother plant pathogen)) sometimes is based on and/or includes an outcome.An outcome and/or classification sometimes is based on and/or includes aresult of data processing for a test sample that facilitates determiningone or more characteristics of a sample (e.g., a Cannabis plant sample)or pathogen (e.g., HpLVd, AMV, BCTV or other plant pathogen) and/orpresence or absence of a genotype, phenotype, genetic variation, geneticalteration, genetic variation signature, and/or infection in aclassification process (e.g., a statistic value). An outcome and/orclassification sometimes includes or is based on a score determinativeof, or a call of, one or more characteristics of a sample (e.g., aCannabis plant sample) or pathogen (e.g., HpLVd, AMV, BCTV or otherplant pathogen) and/or presence or absence of a genotype, phenotype,genetic variation, genetic alteration, genetic variation signature,and/or infection (e.g., an HpLVd, AMV, BCTV or other plant pathogeninfection). In certain embodiments, an outcome and/or classificationincludes a conclusion that predicts and/or determines one or morecharacteristics of a sample (e.g., a Cannabis plant sample) or pathogen(e.g., HpLVd, AMV, BCTV or other plant pathogen) and/or presence orabsence of a genotype, phenotype, genetic variation, genetic alteration,genetic variation signature, and/or infection (e.g., an HpLVd, AMV, BCTVor other plant pathogen infection) in a classification process.

Any suitable expression of an outcome and/or classification can beprovided. An outcome and/or classification sometimes is based on and/orincludes one or more numerical values generated using a processingmethod described herein in the context of one or more considerations ofprobability. Non-limiting examples of values that can be utilizedinclude a sensitivity, specificity, standard deviation, median absolutedeviation (MAD), measure of certainty, measure of confidence, measure ofcertainty or confidence that a value obtained for a test sample isinside or outside a particular range of values, measure of uncertainty,measure of uncertainty that a value obtained for a test sample is insideor outside a particular range of values, coefficient of variation (CV),confidence level, confidence interval (e.g., about 95% confidenceinterval), standard score (e.g., z-score), chi value, phi value, resultof a t-test, p-value, ploidy value, fitted minority species fraction,area ratio, median level, the like or combination thereof. In someembodiments, an outcome and/or classification comprises a read density,a read density profile and/or a plot (e.g., a profile plot). In certainembodiments, multiple values are analyzed together, sometimes in aprofile for such values (e.g., z-score profile, p-value profile, chivalue profile, phi value profile, result of a t-test, value profile, thelike, or combination thereof). A consideration of probability canfacilitate determining one or more characteristics of a sample orpathogen; whether a plant is at risk of having, or has, a genotype,phenotype, genetic variation and/or infection; whether a pathogen has agenotype, genetic variation, or genetic variation signature; and/orwhether a plant has a phenotype associated with a particular pathogenvariant or strain, and an outcome and/or classification determinative ofthe foregoing sometimes includes such a consideration. In someembodiments, a consideration of probability can facilitate determiningone or more characteristics of a Cannabis plant sample or an HpLVd, AMV,BCTV or other plant pathogen variant or strain; whether a cannabis plantis at risk of having, or has, a genotype, phenotype, genetic variationand/or HpLVd, AMV, BCTV or other plant pathogen infection; whether anHpLVd, AMV, BCTV or other plant pathogen variant or strain has agenotype, genetic variation, or genetic variation signature; and/orwhether a cannabis plant has a phenotype associated with a particularHpLVd, AMV, BCTV or other plant pathogen variant or strain, and anoutcome and/or classification determinative of the foregoing sometimesincludes such a consideration.

In certain embodiments, an outcome and/or classification is based onand/or includes a conclusion that predicts and/or determines a risk orprobability of the presence or absence of a genotype, phenotype, geneticvariation and/or infection for a test sample (e.g., a test sample from acannabis plant). In certain embodiments, an outcome and/orclassification is based on and/or includes a conclusion that predictsand/or determines a risk or probability of the presence or absence of agenotype, genetic variation, and/or genetic variation signature apathogen (e.g., HpLVd, AMV, BCTV or other plant pathogen). A conclusionsometimes is based on a value determined from a data analysis methoddescribed herein (e.g., a statistics value indicative of probability,certainty and/or uncertainty (e.g., standard deviation, median absolutedeviation (MAD), measure of certainty, measure of confidence, measure ofcertainty or confidence that a value obtained for a test sample orpathogen is inside or outside a particular range of values, measure ofuncertainty, measure of uncertainty that a value obtained for a testsample or pathogen is inside or outside a particular range of values,coefficient of variation (CV), confidence level, confidence interval(e.g., about 95% confidence interval), standard score (e.g., z-score),chi value, phi value, result of a t-test, p-value, sensitivity,specificity, the like or combination thereof). An outcome and/orclassification sometimes is expressed in a laboratory test report forparticular test sample (e.g., a cannabis plant sample) as a probability(e.g., odds ratio, p-value), likelihood, or risk factor, associated withthe presence or absence of a genotype, phenotype, genetic variationand/or infection. An outcome and/or classification sometimes isexpressed in a laboratory test report for particular pathogen (e.g.,HpLVd, AMV, BCTV or other plant pathogen) as a probability (e.g., oddsratio, p-value), likelihood, or risk factor, associated with thepresence or absence of a genotype, genetic variation, and/or geneticvariation signature. An outcome and/or classification for a test sample(e.g., a Cannabis plant sample) sometimes is provided as “positive” or“negative” with respect a particular genotype, phenotype, geneticvariation and/or infection. For example, an outcome and/orclassification sometimes is designated as “positive” in a laboratorytest report for a particular test sample (e.g., a Cannabis plant sample)where presence of a genotype, phenotype, genetic variation and/orinfection is determined, and sometimes an outcome and/or classificationis designated as “negative” in a laboratory test report for a particulartest sample (e.g., a Cannabis plant sample) where absence of a genotype,phenotype, genetic variation and/or infection is determined. An outcomeand/or classification for a pathogen (e.g., HpLVd, AMV, BCTV or otherplant pathogen) sometimes is provided as “positive” or “negative” withrespect a particular genotype, genetic variation, and/or geneticvariation signature. For example, an outcome and/or classificationsometimes is designated as “positive” in a laboratory test report for aparticular pathogen (e.g., HpLVd, AMV, BCTV or other plant pathogen)where presence of a genotype, genetic variation, and/or geneticvariation signature is determined, and sometimes an outcome and/orclassification is designated as “negative” in a laboratory test reportfor a particular pathogen (e.g., HpLVd, AMV, BCTV or other plantpathogen) where absence of a genotype, genetic variation, and/or geneticvariation signature is determined. An outcome and/or classificationsometimes is determined and sometimes includes an assumption used indata processing.

There typically are four types of classifications generated in aclassification process: true positive, false positive, true negative andfalse negative. The term “true positive” as used herein refers topresence of a genotype, phenotype, genetic variation, or infectioncorrectly determined for a test sample. The term “false positive” asused herein refers to presence of a genotype, phenotype, geneticvariation, or infection incorrectly determined for a test sample. Theterm “true negative” as used herein refers to absence of a genotype,phenotype, genetic variation, or infection correctly determined for atest sample. The term “false negative” as used herein refers to absenceof a genotype, phenotype, genetic variation, or infection incorrectlydetermined for a test sample. Two measures of performance for aclassification process can be calculated based on the ratios of theseoccurrences: (i) a sensitivity value, which generally is the fraction ofpredicted positives that are correctly identified as being positives;and (ii) a specificity value, which generally is the fraction ofpredicted negatives correctly identified as being negative.

In certain embodiments, a laboratory test report generated for aclassification process includes a measure of test performance (e.g.,sensitivity and/or specificity) and/or a measure of confidence (e.g., aconfidence level, confidence interval). A measure of test performanceand/or confidence sometimes is obtained from a clinical validation studyperformed prior to performing a laboratory test for a test sample. Incertain embodiments, one or more of sensitivity, specificity and/orconfidence are expressed as a percentage. In some embodiments, apercentage expressed independently for each of sensitivity, specificityor confidence level, is greater than about 90% (e.g., about 90, 91, 92,93, 94, 95, 96, 97, 98 or 99%, or greater than 99% (e.g., about 99.5%,or greater, about 99.9% or greater, about 99.95% or greater, about99.99% or greater)). A confidence interval expressed for a particularconfidence level (e.g., a confidence level of about 90% to about 99.9%(e.g., about 95%)) can be expressed as a range of values, and sometimesis expressed as a range or sensitivities and/or specificities for aparticular confidence level. Coefficient of variation (CV) in someembodiments is expressed as a percentage, and sometimes the percentageis about 10% or less (e.g., about 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1%, orless than 1% (e.g., about 0.5% or less, about 0.1% or less, about 0.05%or less, about 0.01% or less)). A probability (e.g., that a particularoutcome and/or classification is not due to chance) in certainembodiments is expressed as a standard score (e.g., z-score), a p-value,or result of a t-test. In some embodiments, a measured variance,confidence level, confidence interval, sensitivity, specificity and thelike (e.g., referred to collectively as confidence parameters) for anoutcome and/or classification can be generated using one or more dataprocessing manipulations described herein.

In certain embodiments, an outcome and/or classification is providedusing a suitable visual medium (e.g., a peripheral or component of amachine, e.g., a printer or display). A classification and/or outcomemay be provided in the form of a report. A report typically comprises adisplay of an outcome and/or classification (e.g., a value, one or morecharacteristics of a sample or pathogen, an assessment or probability ofpresence or absence of a genotype, phenotype, genetic variation and/orinfection; and/or an assessment or probability of a genotype, geneticvariation, and/or genetic variation signature for a pathogen), sometimesincludes an associated confidence parameter, and sometimes includes ameasure of performance for a test used to generate the outcome and/orclassification. A report sometimes includes a recommendation for afollow-up test (e.g., a test that confirms the outcome orclassification).

A report can be displayed in a suitable format that facilitatesdetermination of presence or absence of a genotype, phenotype, geneticvariation, genetic variation signature, and/or infection. Non-limitingexamples of formats suitable for use for generating a report includedigital data, a graph, a 2D graph, a 3D graph, and 4D graph, a picture(e.g., a jpg, bitmap (e.g., bmp), pdf, tiff, gif, raw, png, the like orsuitable format), a pictograph, a chart, a table, a bar graph, a piegraph, a diagram, a flow chart, a scatter plot, a map, a histogram, adensity chart, a function graph, a circuit diagram, a block diagram, abubble map, a constellation diagram, a contour diagram, a cartogram,spider chart, Venn diagram, nomogram, and the like, or combination ofthe foregoing.

A report may be generated by a computer and/or by human data entry, andcan be transmitted and communicated using a suitable electronic medium(e.g., via the internet, via computer, via facsimile, from one networklocation to another location at the same or different physical sites),or by another method of sending or receiving data (e.g., mail service,courier service and the like). Non-limiting examples of communicationmedia for transmitting a report include auditory file, computer readablefile (e.g., pdf file), paper file, laboratory file, or any other mediumdescribed in the previous paragraph. A laboratory file may be intangible form or electronic form (e.g., computer readable form), incertain embodiments. After a report is generated and transmitted, areport can be received by obtaining, via a suitable communicationmedium, a written and/or graphical representation comprising an outcomeand/or classification, which upon review allows a qualified individualto make a determination as to one or more characteristics of a sample orpathogen; presence or absence of a genotype, phenotype, geneticvariation and/or infection for a test sample (e.g., a Cannabis plantsample); and/or genotype, genetic variation, and/or genetic variationsignature for a pathogen (e.g., HpLVd, AMV, BCTV or other plantpathogen).

An outcome and/or classification may be provided by and obtained from alaboratory (e.g., obtained from a laboratory file). A laboratory filecan be generated by a laboratory that carries out one or more tests fordetermining one or more characteristics of a sample or pathogen;presence or absence of a genotype, phenotype, genetic variation and/orinfection for a test sample (e.g., a cannabis plant sample); and/orgenotype, genetic variation, and/or genetic variation signature for apathogen (e.g., HpLVd, AMV, BCTV or other plant pathogen). Laboratorypersonnel (e.g., a laboratory manager) can analyze informationassociated with test samples (e.g., test profiles, reference profiles,test values, reference values, level of deviation) underlying an outcomeand/or classification. For calls pertaining to presence or absence of agenotype, phenotype, genetic variation and/or infection that are closeor questionable, laboratory personnel can re-run the same procedureusing the same (e.g., aliquot of the same sample) or different testsample from a plant. A laboratory may be in the same location ordifferent location (e.g., in another country) as personnel assessing thepresence or absence of a genotype, phenotype, genetic variation and/orinfection from the laboratory file. For example, a laboratory file canbe generated in one location and transmitted to another location inwhich the information for a test sample therein is assessed by aqualified individual, and optionally, transmitted to the facility and/orgrower from which the test sample was obtained. A laboratory sometimesgenerates and/or transmits a laboratory report containing aclassification of presence or absence of a genotype, phenotype, agenetic variation, and/or an infection for a test sample (e.g., aCannabis plant sample); and/or a genotype, genetic variation, and/orgenetic variation signature for a pathogen (e.g., HpLVd, AMV, BCTV orother plant pathogen).

An outcome and/or classification sometimes is a component of a diagnosisfor a plant, and sometimes an outcome and/or classification is utilizedand/or assessed as part of providing a diagnosis for a test sample. Forexample, a qualified individual may analyze an outcome and/orclassification and provide a diagnosis based on, or based in part on,the outcome and/or classification. In some embodiments, determination,detection or diagnosis of an infection, disease, and/or abnormalitycomprises use of an outcome and/or classification determinative ofpresence or absence of a genotype, phenotype, genetic variation and/orinfection for a test sample (e.g., a Cannabis plant sample); and/or agenotype, genetic variation, and/or genetic variation signature for apathogen (e.g., HpLVd, AMV, BCTV or other plant pathogen). Thus,provided herein are methods for diagnosing presence or absence of agenotype, phenotype, a genetic variation and/or an infection for a testsample (e.g., a Cannabis plant sample) according to an outcome orclassification generated by methods described herein, and optionallyaccording to generating and transmitting a laboratory report thatincludes a classification for presence or absence of the genotype,phenotype, a genetic variation and/or an infection for the test sample(e.g., a Cannabis plant sample). Also provided herein are methods fordiagnosing presence or absence of a genotype, phenotype, a geneticvariation and/or an infection for a test sample (e.g., a Cannabis plantsample) according to an outcome or classification generated by methodsdescribed herein for a genotype, genetic variation, and/or geneticvariation signature for a pathogen (e.g., HpLVd, AMV, BCTV or otherplant pathogen), and optionally according to generating and transmittinga laboratory report that includes a classification for presence orabsence of the genotype, phenotype, a genetic variation and/or aninfection for the test sample (e.g., a cannabis plant sample), and/or aclassification of a genotype, genetic variation, and/or geneticvariation signature for a pathogen (e.g., HpLVd, AMV, BCTV or otherplant pathogen).

Machines, Software and Interfaces

Methods described herein (e.g., processing amplification results,processing high resolution melting (HRM) assay results, processingsequence read data, determining one or more characteristics of a sampleor a pathogen based on sequence read data, associating one or morephenotypes of an infected plant (e.g., an infected cannabis plant) withone or more genotypes, genetic variations, and/or genetic variationsignatures for a pathogen (e.g., HpLVd, AMV, BCTV or other plantpathogen), and/or providing an outcome) may be computer-implementedmethods, and one or more portions of a method sometimes are performed byone or more processors (e.g., microprocessors), computers, systems,apparatuses, or machines (e.g., microprocessor-controlled machine).

Computers, systems, apparatuses, machines and computer program productssuitable for use often include, or are utilized in conjunction with,computer readable storage media. Non-limiting examples of computerreadable storage media include memory, hard disk, CD-ROM, flash memorydevice and the like. Computer readable storage media generally arecomputer hardware, and often are non-transitory computer-readablestorage media. Computer readable storage media are not computer readabletransmission media, the latter of which are transmission signals per se.

Provided herein are computer readable storage media with an executableprogram stored thereon, where the program instructs a microprocessor toperform a method described herein. Provided also are computer readablestorage media with an executable program module stored thereon, wherethe program module instructs a microprocessor to perform part of amethod described herein. Also provided herein are systems, machines,apparatuses and computer program products that include computer readablestorage media with an executable program stored thereon, where theprogram instructs a microprocessor to perform a method described herein.Provided also are systems, machines and apparatuses that includecomputer readable storage media with an executable program module storedthereon, where the program module instructs a microprocessor to performpart of a method described herein.

Also provided are computer program products. A computer program productoften includes a computer usable medium that includes a computerreadable program code embodied therein, the computer readable programcode adapted for being executed to implement a method, or part of amethod, described herein. Computer usable media and readable programcode are not transmission media (i.e., transmission signals per se).Computer readable program code often is adapted for being executed by aprocessor, computer, system, apparatus, or machine.

In some embodiments, methods described herein (e.g., processingamplification results, processing high resolution melting (HRM) assayresults, processing sequence read data, determining one or morecharacteristics of a sample or a pathogen based on sequence read data,associating one or more phenotypes of an infected plant (e.g., aninfected Cannabis plant) with one or more genotypes, genetic variations,and/or genetic variation signatures for a pathogen (e.g., HpLVd, AMV,BCTV or other plant pathogen), and/or providing an outcome) areperformed by automated methods. In some embodiments, one or more stepsof a method described herein are carried out by a microprocessor and/orcomputer, and/or carried out in conjunction with memory. In someembodiments, an automated method is embodied in software, modules,microprocessors, peripherals and/or a machine comprising the like, thatperform methods described herein. As used herein, software refers tocomputer readable program instructions that, when executed by amicroprocessor, perform computer operations, as described herein.

Machines, software and interfaces may be used to conduct methodsdescribed herein. Using machines, software and interfaces, a user mayenter, request, query or determine options for using particularinformation, programs or processes (e.g., processing amplificationresults, processing high resolution melting (HRM) assay results,processing sequence read data, determining one or more characteristicsof a sample or a pathogen based on sequence read data, associating oneor more phenotypes of an infected plant (e.g., an infected cannabisplant) with one or more genotypes, genetic variations, and/or geneticvariation signatures for a pathogen (e.g., HpLVd, AMV, BCTV or otherplant pathogen), and/or providing an outcome), which can involveimplementing statistical analysis algorithms, statistical significancealgorithms, statistical algorithms, iterative steps, validationalgorithms, and graphical representations, for example. In someembodiments, a data set may be entered by a user as input information, auser may download one or more data sets by suitable hardware media(e.g., flash drive), and/or a user may send a data set from one systemto another for subsequent processing and/or providing an outcome (e.g.,send sequence read data from a sequencer to a computer system forsequence read processing; send processed sequence read data to acomputer system for further processing and/or yielding an outcome and/orreport).

A system typically comprises one or more machines. Each machinecomprises one or more of memory, one or more microprocessors, andinstructions. Where a system includes two or more machines, some or allof the machines may be located at the same location, some or all of themachines may be located at different locations, all of the machines maybe located at one location and/or all of the machines may be located atdifferent locations. Where a system includes two or more machines, someor all of the machines may be located at the same location as a user,some or all of the machines may be located at a location different thana user, all of the machines may be located at the same location as theuser, and/or all of the machine may be located at one or more locationsdifferent than the user.

A system sometimes comprises a computing machine and a sequencingapparatus or machine, where the sequencing apparatus or machine isconfigured to receive physical nucleic acid and generate sequence reads,and the computing apparatus is configured to process the reads from thesequencing apparatus or machine. The computing machine sometimes isconfigured to determine an outcome from the sequence reads.

A user may, for example, place a query to software which then mayacquire a data set via internet access, and in certain embodiments, aprogrammable microprocessor may be prompted to acquire a suitable dataset based on given parameters. A programmable microprocessor also mayprompt a user to select one or more data set options selected by themicroprocessor based on given parameters. A programmable microprocessormay prompt a user to select one or more data set options selected by themicroprocessor based on information found via the internet, otherinternal or external information, or the like. Options may be chosen forselecting one or more data feature selections, one or more statisticalalgorithms, one or more statistical analysis algorithms, one or morestatistical significance algorithms, iterative steps, one or morevalidation algorithms, and one or more graphical representations ofmethods, machines, apparatuses, computer programs or a non-transitorycomputer-readable storage medium with an executable program storedthereon.

Systems addressed herein may comprise general components of computersystems, such as, for example, network servers, laptop systems, desktopsystems, handheld systems, personal digital assistants, computingkiosks, and the like. A computer system may comprise one or more inputmeans such as a keyboard, touch screen, mouse, voice recognition orother means to allow the user to enter data into the system. A systemmay further comprise one or more outputs, including, but not limited to,a display screen (e.g., CRT or LCD), speaker, FAX machine, printer(e.g., laser, ink jet, impact, black and white or color printer), orother output useful for providing visual, auditory and/or hardcopyoutput of information (e.g., outcome and/or report).

In a system, input and output components may be connected to a centralprocessing unit which may comprise among other components, amicroprocessor for executing program instructions and memory for storingprogram code and data. In some embodiments, processes may be implementedas a single user system located in a single geographical site. Incertain embodiments, processes may be implemented as a multi-usersystem. In the case of a multi-user implementation, multiple centralprocessing units may be connected by means of a network. The network maybe local, encompassing a single department in one portion of a building,an entire building, span multiple buildings, span a region, span anentire country or be worldwide. The network may be private, being ownedand controlled by a provider, or it may be implemented as an internetbased service where the user accesses a web page to enter and retrieveinformation. Accordingly, in certain embodiments, a system includes oneor more machines, which may be local or remote with respect to a user.More than one machine in one location or multiple locations may beaccessed by a user, and data may be mapped and/or processed in seriesand/or in parallel. Thus, a suitable configuration and control may beutilized for mapping and/or processing data using multiple machines,such as in local network, remote network and/or “cloud” computingplatforms.

A system can include a communications interface in some embodiments. Acommunications interface allows for transfer of software and databetween a computer system and one or more external devices. Non-limitingexamples of communications interfaces include a modem, a networkinterface (such as an Ethernet card), a communications port, a PCMCIAslot and card, and the like. Software and data transferred via acommunications interface generally are in the form of signals, which canbe electronic, electromagnetic, optical and/or other signals capable ofbeing received by a communications interface. Signals often are providedto a communications interface via a channel. A channel often carriessignals and can be implemented using wire or cable, fiber optics, aphone line, a cellular phone link, an RF link and/or othercommunications channels. Thus, in an example, a communications interfacemay be used to receive signal information that can be detected by asignal detection module.

Data may be input by a suitable device and/or method, including, but notlimited to, manual input devices or direct data entry devices (DDEs).Non-limiting examples of manual devices include keyboards, conceptkeyboards, touch sensitive screens, light pens, mouse, tracker balls,joysticks, graphic tablets, scanners, digital cameras, video digitizersand voice recognition devices. Non-limiting examples of DDEs include barcode readers, magnetic strip codes, smart cards, magnetic ink characterrecognition, optical character recognition, optical mark recognition,and turnaround documents.

A system may include software useful for performing a process or part ofa process described herein, and software can include one or more modulesfor performing such processes (e.g., sequencing module, logic processingmodule, data display organization module). The term “software” refers tocomputer readable program instructions that, when executed by acomputer, perform computer operations. Instructions executable by theone or more microprocessors sometimes are provided as executable code,that when executed, can cause one or more microprocessors to implement amethod described herein. A module described herein can exist assoftware, and instructions (e.g., processes, routines, subroutines)embodied in the software can be implemented or performed by amicroprocessor. For example, a module (e.g., a software module) can be apart of a program that performs a particular process or task. The term“module” refers to a self-contained functional unit that can be used ina larger machine or software system. A module can comprise a set ofinstructions for carrying out a function of the module. A module cantransform data and/or information. Data and/or information can be in asuitable form. For example, data and/or information can be digital oranalogue. In certain embodiments, data and/or information sometimes canbe packets, bytes, characters, or bits. In some embodiments, data and/orinformation can be any gathered, assembled or usable data orinformation. Non-limiting examples of data and/or information include asuitable media, pictures, video, sound (e.g., frequencies, audible ornon-audible), numbers, constants, a value, objects, time, functions,instructions, maps, references, sequences, reads, mapped reads, levels,ranges, thresholds, signals, displays, representations, ortransformations thereof. A module can accept or receive data and/orinformation, transform the data and/or information into a second form,and provide or transfer the second form to a machine, peripheral,component or another module. A microprocessor can, in certainembodiments, carry out the instructions in a module. In someembodiments, one or more microprocessors are required to carry outinstructions in a module or group of modules. A module can provide dataand/or information to another module, machine or source and can receivedata and/or information from another module, machine or source.

A computer program product sometimes is embodied on a tangiblecomputer-readable medium, and sometimes is tangibly embodied on anon-transitory computer-readable medium. A module sometimes is stored ona computer readable medium (e.g., disk, drive) or in memory (e.g.,random access memory). A module and microprocessor capable ofimplementing instructions from a module can be located in a machine orin a different machine. A module and/or microprocessor capable ofimplementing an instruction for a module can be located in the samelocation as a user (e.g., local network) or in a different location froma user (e.g., remote network, cloud system). In embodiments in which amethod is carried out in conjunction with two or more modules, themodules can be located in the same machine, one or more modules can belocated in different machine in the same physical location, and one ormore modules may be located in different machines in different physicallocations.

A machine, in some embodiments, comprises at least one microprocessorfor carrying out the instructions in a module. In some embodiments, amachine includes a microprocessor (e.g., one or more microprocessors)which microprocessor can perform and/or implement one or moreinstructions (e.g., processes, routines and/or subroutines) from amodule. In some embodiments, a machine includes multiplemicroprocessors, such as microprocessors coordinated and working inparallel. In some embodiments, a machine operates with one or moreexternal microprocessors (e.g., an internal or external network, server,storage device and/or storage network (e.g., a cloud)). In someembodiments, a machine comprises a module (e.g., one or more modules). Amachine comprising a module often is capable of receiving andtransferring one or more of data and/or information to and from othermodules.

In certain embodiments, a machine comprises peripherals and/orcomponents. In certain embodiments, a machine can comprise one or moreperipherals or components that can transfer data and/or information toand from other modules, peripherals and/or components. In certainembodiments, a machine interacts with a peripheral and/or component thatprovides data and/or information. In certain embodiments, peripheralsand components assist a machine in carrying out a function or interactdirectly with a module. Non-limiting examples of peripherals and/orcomponents include a suitable computer peripheral, I/O or storage methodor device including but not limited to scanners, printers, displays(e.g., monitors, LED, LCT or CRTs), cameras, microphones, pads (e.g.,ipads, tablets), touch screens, smart phones, mobile phones, USB I/Odevices, USB mass storage devices, keyboards, a computer mouse, digitalpens, modems, hard drives, jump drives, flash drives, a microprocessor,a server, CDs, DVDs, graphic cards, specialized I/O devices (e.g.,sequencers, photo cells, photo multiplier tubes, optical readers,sensors, etc.), one or more flow cells, fluid handling components,network interface controllers, ROM, RAM, wireless transfer methods anddevices (Bluetooth, WiFi, and the like), the world wide web (www), theinternet, a computer and/or another module.

Software often is provided on a program product containing programinstructions recorded on a computer readable medium, including, but notlimited to, magnetic media including floppy disks, hard disks, andmagnetic tape; and optical media including CD-ROM discs, DVD discs,magneto-optical discs, flash memory devices (e.g., flash drives), RAM,floppy discs, the like, and other such media on which the programinstructions can be recorded. In online implementation, a server and website maintained by an organization can be configured to provide softwaredownloads to remote users, or remote users may access a remote systemmaintained by an organization to remotely access software. Software mayobtain or receive input information. Software may include a module thatspecifically obtains or receives data and may include a module thatspecifically processes the data (e.g., a processing module thatprocesses received data). The terms “obtaining” and “receiving” inputinformation refers to receiving data by computer communication meansfrom a local, or remote site, human data entry, or any other method ofreceiving data. The input information may be generated in the samelocation at which it is received, or it may be generated in a differentlocation and transmitted to the receiving location. In some embodiments,input information is modified before it is processed (e.g., placed intoa format amenable to processing (e.g., tabulated)).

Software can include one or more algorithms in certain embodiments. Analgorithm may be used for processing data and/or providing an outcome orreport according to a finite sequence of instructions. An algorithmoften is a list of defined instructions for completing a task. Startingfrom an initial state, the instructions may describe a computation thatproceeds through a defined series of successive states, eventuallyterminating in a final ending state. The transition from one state tothe next is not necessarily deterministic (e.g., some algorithmsincorporate randomness). By way of example, and without limitation, analgorithm can be a search algorithm, sorting algorithm, merge algorithm,numerical algorithm, graph algorithm, string algorithm, modelingalgorithm, computational genometric algorithm, combinatorial algorithm,machine learning algorithm, cryptography algorithm, data compressionalgorithm, parsing algorithm and the like. An algorithm can include onealgorithm or two or more algorithms working in combination. An algorithmcan be of any suitable complexity class and/or parameterized complexity.An algorithm can be used for calculation and/or data processing, and insome embodiments, can be used in a deterministic orprobabilistic/predictive approach. An algorithm can be implemented in acomputing environment by use of a suitable programming language,non-limiting examples of which are C, C++, Java, Perl, Python, Fortran,and the like. In some embodiments, an algorithm can be configured ormodified to include margin of errors, statistical analysis, statisticalsignificance, and/or comparison to other information or data sets (e.g.,applicable when using a neural net or clustering algorithm).

In certain embodiments, several algorithms may be implemented for use insoftware. These algorithms can be trained with raw data in someembodiments. For each new raw data sample, the trained algorithms mayproduce a representative processed data set or outcome. A processed dataset sometimes is of reduced complexity compared to the parent data setthat was processed. Based on a processed set, the performance of atrained algorithm may be assessed based on sensitivity and specificity,in some embodiments. An algorithm with the highest sensitivity and/orspecificity may be identified and utilized, in certain embodiments.

In certain embodiments, simulated (or simulation) data can aid dataprocessing, for example, by training an algorithm or testing analgorithm. In some embodiments, simulated data includes hypotheticalvarious samplings of different groupings of sequence reads, genotypes,phenotypes, genetic variations, and/or genetic variation signatures.Simulated data may be based on what might be expected from a realpopulation or may be skewed to test an algorithm and/or to assign acorrect classification. Simulated data also is referred to herein as“virtual” data. Simulations can be performed by a computer program incertain embodiments. One possible step in using a simulated data set isto evaluate the confidence of identified results, e.g., how well arandom sampling matches or best represents the original data. Oneapproach is to calculate a probability value (p-value), which estimatesthe probability of a random sample having better score than the selectedsamples. In some embodiments, an empirical model may be assessed, inwhich it is assumed that at least one sample matches a reference sample(with or without resolved variations). In some embodiments, anotherdistribution, such as a Poisson distribution for example, can be used todefine the probability distribution.

A system may include one or more microprocessors in certain embodiments.A microprocessor can be connected to a communication bus. A computersystem may include a main memory, often random access memory (RAM), andcan also include a secondary memory. Memory in some embodimentscomprises a non-transitory computer-readable storage medium. Secondarymemory can include, for example, a hard disk drive and/or a removablestorage drive, representing a floppy disk drive, a magnetic tape drive,an optical disk drive, memory card and the like. A removable storagedrive often reads from and/or writes to a removable storage unit.Non-limiting examples of removable storage units include a floppy disk,magnetic tape, optical disk, and the like, which can be read by andwritten to by, for example, a removable storage drive. A removablestorage unit can include a computer-usable storage medium having storedtherein computer software and/or data.

A microprocessor may implement software in a system. In someembodiments, a microprocessor may be programmed to automatically performa task described herein that a user could perform. Accordingly, amicroprocessor, or algorithm conducted by such a microprocessor, canrequire little to no supervision or input from a user (e.g., softwaremay be programmed to implement a function automatically). In someembodiments, the complexity of a process is so large that a singleperson or group of persons could not perform the process in a timeframeshort enough for determining one or more characteristics of a sample.

In some embodiments, secondary memory may include other similar meansfor allowing computer programs or other instructions to be loaded into acomputer system. For example, a system can include a removable storageunit and an interface device. Non-limiting examples of such systemsinclude a program cartridge and cartridge interface (such as that foundin video game devices), a removable memory chip (such as an EPROM, orPROM) and associated socket, and other removable storage units andinterfaces that allow software and data to be transferred from theremovable storage unit to a computer system.

Compositions

Provided in certain embodiments are compositions. Compositions usefulfor carrying out any of the methods described herein are provided. Forexample, compositions comprising any of the primers, primer pairs,primer sets, probes, and/or reverse complements thereof described hereinare provided.

In some embodiments, a composition comprises one or more polynucleotideprimer pairs (e.g., one or more polynucleotide primer pairs describedherein). In some embodiments, each polynucleotide of the one or moreprimer pairs is identical, or substantially identical, to a subsequenceof SEQ ID NO:1, or complement thereof. In some embodiments, eachsubsequence of SEQ ID NO:1, or complement thereof, to which eachpolynucleotide is identical, or substantially identical, contains novariant nucleotide position. In some embodiments, each target sequenceof SEQ ID NO:1 between the subsequences, or complements thereof, towhich the polynucleotides of the one or more primer pairs are identical,or substantially identical, (i.e., the subsequence between the primerhybridization sites) comprises one or more variant nucleotide positions.

In some embodiments, a composition comprises one or more furtherpolynucleotide primers. In some embodiments, each polynucleotide of theone or more further polynucleotide primers is identical, orsubstantially identical, to a subsequence of SEQ ID NO:1, or complementthereof. In some embodiments, each subsequence of SEQ ID NO:1, orcomplement thereof, to which each polynucleotide is identical, orsubstantially identical, contains one or more variant nucleotidepositions.

In some embodiments, a composition comprises a) a first set ofpolynucleotide primers where i) each polynucleotide of the a first setof polynucleotide primers is identical, or substantially identical, to asubsequence of SEQ ID NO:1, or complement thereof, ii) each subsequenceof SEQ ID NO:1, or complement thereof, to which each polynucleotide isidentical, or substantially identical, contains no variant nucleotideposition, and iii) each target sequence of SEQ ID NO:1 between thesubsequences, or complements thereof, to which the polynucleotides ofthe first set of polynucleotide primers are identical, or substantiallyidentical, comprises one or more variant nucleotide positions; and b) asecond set of polynucleotide primers where i) each polynucleotide of thesecond set of polynucleotide primers is identical, or substantiallyidentical, to a subsequence of SEQ ID NO:1, or complement thereof, andii) each subsequence of SEQ ID NO:1, or complement thereof, to whicheach polynucleotide is identical, or substantially identical, containsone or more variant nucleotide positions.

In some embodiments, a composition comprises at least one polynucleotideprimer pair that is capable of specifically hybridizing to andamplifying a subsequence of the nucleic acid of Alfalfa Mosaic Virus(AMV). In certain embodiments, the subsequence of the nucleic acid ofthe Alfalfa Mosaic Virus (AMV) to which the polynucleotide primer pairis capable of hybridizing comprises SEQ ID NO:91, or a portion of SEQ IDNO:91, or a complement of SEQ ID NO:91, or a portion of the complementof SEQ ID NO:91. In embodiments, the at least one polynucleotide primerpair is selected from among: one primer selected from among those havingthe sequences set forth in SEQ ID NOS: 80, 82 and 85, or from amongsequences that share 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ormore identity with the sequences set forth in SEQ ID NOS: 80, 82 and 85;and one primer selected from among those having the sequences set forthin SEQ ID NOS: 81, 83, 84 and 86; or from among sequences that share90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity withthe sequences set forth in SEQ ID NOS: 81, 83, 84 and 86.

In some embodiments, a composition comprises at least one polynucleotideprimer pair that is capable of specifically hybridizing to andamplifying a subsequence of the nucleic acid of Beet Curly Top Virus(BCTV). In certain embodiments, the subsequence of the nucleic acid ofthe pathogen to which the at least one polynucleotide primer pair iscapable of hybridizing is selected from among SEQ ID NOS:110, 112, 114,116, 118 or 120, or a portion of SEQ ID NOS:110, 112, 114, 116, 118 or120, or a complement of SEQ ID NOS:110, 112, 114, 116, 118 or 120, or aportion of the complement of SEQ ID NOS:110, 112, 114, 116, 118 or 120,or to regions of overlap that span any two of SEQ ID NOS:110, 112, 114,116, 118 or 120 in the genome of the pathogen. I embodiments, thesubsequence of the nucleic acid of the pathogen to which the at leastone polynucleotide primer pair is capable of hybridizing is in a regionof overlap that spans:

-   -   (i) the gene encoding the SS-ds-DNA Regulator Protein (SEQ ID        NO:110) and the gene encoding Movement Protein (SEQ ID NO:112);    -   (ii) the gene encoding the Pathogenesis Enhancement Protein (SEQ        ID NO:116) and the gene encoding the Rolling Circle Replication        Protein (SEQ ID NO:114);    -   (iii) the gene encoding the Rolling Circle Replication Protein        (SEQ ID NO:114) and the gene encoding the Cell Cycle Regulator        Protein (SEQ ID NO:118); or    -   (iv) the gene encoding the Pathogenesis Enhancement Protein (SEQ        ID NO:116) and the gene encoding the Replication Enhancer        Protein (SEQ ID NO:120). In certain embodiments, the        polynucleotide primer pairs comprise:    -   for (i), the primer pair having the sequences set forth in SEQ        ID NOS: 93 and 94 or sequences that share 90%, 91%, 92%, 93%,        94%, 95%, 96%, 97%, 98%, 99% or more identity with the sequences        set forth in SEQ ID NOS: 93 and 94, or the primer pair having        the sequences set forth in SEQ ID NOS: 93 and 105, or sequences        that share 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or        more identity with the sequences set forth in SEQ ID NOS: 93 and        105;    -   for (ii), the primers having the sequences set forth in SEQ ID        NOS: 96 and 97, or sequences that share 90%, 91%, 92%, 93%, 94%,        95%, 96%, 97%, 98%, 99% or more identity with the sequences set        forth in SEQ ID NOS: 96 and 97;    -   for (iii), the primers having the sequences set forth in SEQ ID        NOS: 99 and 100, or sequences that share 90%, 91%, 92%, 93%,        94%, 95%, 96%, 97%, 98%, 99% or more identity with the sequences        set forth in SEQ ID NOS: 99 and 100; and    -   for (iv), the primers having the sequences set forth in SEQ ID        NOS: 102 and 103, or sequences that share 90%, 91%, 92%, 93%,        94%, 95%, 96%, 97%, 98%, 99% or more identity with the sequences        set forth in SEQ ID NOS: 102 and 103.

Any of the compositions provided herein can further include one or morepolynucleotide probes as provided herein, for quantifying ampliconsgenerated by the polynucleotide primer pairs of the compositionsprovided herein.

Kits

Provided in certain embodiments are kits. The kits may include anycomponents and compositions described herein (e.g., primers, primerpairs, primer sets (e.g., one or more LAMP primer sets), probes, and/orreverse complements thereof) useful for performing any of the methodsdescribed herein, in any suitable combination. Kits may further includeany reagents, buffers, or other components useful for carrying out anyof the methods described herein. For example, a kit may include one ormore primer pairs described herein and one or more components foramplifying nucleic acid.

Kits may include components for amplifying nucleic acid. Kits foramplifying nucleic acid may be configured such that a user provides aDNA template (e.g., a cDNA template) or an RNA template. A kit foramplifying nucleic acid from an RNA template may further includereagents for reverse transcription (i.e., for generating cDNA).

Components of a kit may be present in separate containers, or multiplecomponents may be present in a single container. In some embodiments,primers are provided such that each container contains a single primerpair (e.g., for individual amplification reactions). In someembodiments, primers are provided such that one container contains aplurality of primer pairs (e.g., for multiplex amplification reactions).Suitable containers include a single tube (e.g., vial), one or morewells of a plate (e.g., a 96-well plate, a 384-well plate, and thelike), and the like.

Kits may also comprise instructions for performing one or more methodsdescribed herein and/or a description of one or more componentsdescribed herein. For example, a kit may include instructions for usingthe amplification primers and/or probes described herein, to amplifynucleic acid (e.g., to amplify subsequences of an HpLVd, AMV, BCTV orother plant pathogen genome). In certain configurations, a kit mayinclude instructions or a guide for interpreting the results of anamplification reaction. Instructions and/or descriptions may be inprinted form and may be included in a kit insert. In some embodiments,instructions and/or descriptions are provided as an electronic storagedata file present on a suitable computer readable storage medium, e.g.,portable flash drive, DVD, CD-ROM, diskette, and the like. A kit alsomay include a written description of an internet location that providessuch instructions or descriptions.

Solid Supports

Provided herein are solid supports that include nucleic acid from aplant sample and any of the polynucleotide primers provided herein. Thenucleic acid and/or primers can directly be attached to the solidsupport, such as by covalent linkage, or can otherwise be associatedwith the solid support. For example, the primers can include, inaddition to a sequence complementary to a unique subsequence of nucleicacid of the genome of a plant cultivar of interest, a sequence that iscomplementary to a nucleic acid sequence that is directly attached tothe solid support. The solid supports that include the primers providedherein can be contacted with nucleic acid from a sample obtained from aplant cultivar, under conditions that facilitate hybridization of aprimer to a corresponding subsequence of the genome of a plant pathogenthat may have infected a plant cultivar of interest. The resultinghybrids can directly be analyzed, such as by a signal or a label, forthe presence or absence of hybridized product containing one or moreprimers specifically bound to a unique subsequence of a pathogen in thenucleic acid of a plant sample. Alternately, the resulting hybrids canbe subjected to polymerase-based amplification reaction conditionsusing, e.g., one or more labeled nucleotides that can be incorporatedinto an amplicon thereby identifying, based on the presence or absenceof a label in the amplicon, whether or not a plant pathogen is plantcultivar of interest.

The term “solid support” or “solid phase” as used herein refers to awide variety of materials including solids, semi-solids, gels, films,membranes, meshes, felts, composites, particles, and the like typicallyused to sequester molecules, and more specifically refers to aninsoluble material with which nucleic acid can be associated. A solidsupport for use with processes described herein sometimes is selected inpart according to size: solid supports having a size smaller than thesize a microreactor sometimes are selected. Examples of solid supportsfor use with processes described herein include, without limitation,beads (e.g., microbeads, nanobeads), particles (e.g., microparticles,nanoparticles) and chips.

The terms “beads” and “particles” as used herein refer to solid supportssuitable for associating with biomolecules, and more specificallynucleic acids. Beads may have a regular (e.g., spheroid, ovoid) orirregular shape (e.g., rough, jagged), and sometimes are non-spherical(e.g., angular, multi-sided). Particles or beads having a nominal,average or mean diameter less than the nominal, average, mean or minimumdiameter of a microreactor can be utilized. Particles or beads having anominal, average or mean diameter of about 1 nanometer to about 500micrometers can be utilized, such as those having a nominal, mean oraverage diameter, for example, of about 10 nanometers to about 100micrometers; about 100 nanometers to about 100 micrometers; about 1micrometer to about 100 micrometers; about 10 micrometers to about 50micrometers; about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65,70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800 or 900nanometers; or about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60,65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500 micrometers.

A bead or particle can be made of virtually any insoluble or solidmaterial. For example, the bead or particle can comprise or consistessentially of silica gel, glass (e.g., controlled-pore glass (CPG)),nylon, Sephadex®, Sepharose®, cellulose, a metal surface (e.g., steel,gold, silver, aluminum, silicon and copper), a magnetic material, aplastic material (e.g., polyethylene, polypropylene, polyamide,polyester, polyvinylidenedifluoride (PVDF)) and the like. Beads orparticles may be swellable (e.g., polymeric beads such as Wang resin) ornon-swellable (e.g., CPG). Commercially available examples of beadsinclude without limitation Wang resin, Merrifield resin and Dynabeads®.Beads may also be made as solid particles or particles that containinternal voids.

The solid supports can be provided in a collection of solid supports. Asolid support collection can include two or more different solid supportspecies. The term “solid support species” as used herein refers to asolid support in association with one particular primer or primer pairprovided herein, or a combination of different primers or primer pairs.In certain embodiments, a solid support includes about 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85,90, 95, 100, 200, 300, 400, 500, 600, 650 or 700 or more primers thatspecifically bind to unique subsequences of one or more TPS genes orparalogs thereof in one or more plant cultivars of interest. The solidsupports (e.g., beads) in the collection of solid supports can behomogeneous (e.g., all are Wang resin beads) or heterogeneous (e.g.,some are Wang resin beads, and some are magnetic beads).

The primers generally are single-stranded and are of any type suitablefor hybridizing sample nucleic acid (e.g., DNA, RNA, analogs thereof(e.g., peptide nucleic acid (PNA)), chimeras thereof (e.g., a singlestrand comprises RNA bases and DNA bases) and the like). The primers ornucleic acid from the plant cultivar sample can be associated with thesolid support in any manner suitable for hybridization of the primers tonucleic acid from the plant cultivar. The primers or nucleic acid fromthe plant cultivar sample can be in association with a solid support bya covalent linkage or a non-covalent interaction. Non-limiting examplesof non-covalent interactions include hydrophobic interactions (e.g., C18coated solid support and tritylated nucleic acid), polar interactions(e.g., “wetting” association between nucleic acid/polyethylene glycol),pair interactions including without limitation, antibody/antigen,antibody/antibody, antibody/antibody fragment, antibody/antibodyreceptor, antibody/protein A or protein G, hapten/anti-hapten,biotin/avidin, biotin/streptavidin, folic acid/folate binding protein,vitamin B12/intrinsic factor, nucleic acid/complementary nucleic acid(e.g., DNA, RNA, PNA) and the like.

The primers provided herein also can be associated with a solid supportby different methodology, which include, without limitation (i)sequentially synthesizing nucleic acid directly on a solid support, and(ii) synthesizing nucleic acid, providing the nucleic acid in solutionphase and linking the nucleic acid to a solid support. The primers canbe linked covalently at various sites in the nucleic acid to the solidsupport, such as (i) at a 1′, 2′, 3′, 4′ or 5′ position of a sugarmoiety or (ii) a pyrimidine or purine base moiety, of a terminal ornon-terminal nucleotide of the nucleic acid, for example. The 5′terminal nucleotide of the primer can be linked to the solid support, incertain embodiments.

Methods for sequentially synthesizing nucleic acid directly on a solidsupport are known. For example, the 3′ end of nucleic acid can be linkedto the solid support (e.g., phosphoramidite method described inCaruthers, Science 230: 281-286 (1985)) or the 5′ end of the nucleicacid can be linked to the solid support (e.g., Claeboe et al, NucleicAcids Res. 31(19): 5685-5691 (2003)).

Methods for linking solution phase nucleic acid to a solid support alsoare known (e.g., U.S. Pat. No. 6,133,436, naming Koster et al. andentitled “Beads bound to a solid support and to nucleic acids” and WO91/08307, naming Van Ness and entitled “Enhanced capture of targetnucleic acid by the use of oligonucleotides covalently attached topolymers”). Examples include, without limitation, thioether linkages(e.g., thiolated nucleic acid); disulfide linkages (e.g., thiol beads,thiolated nucleic acid); amide linkages (e.g., Wang resin, amino-linkednucleic acid); acid labile linkages (e.g., glass beads, tritylatednucleic acid) and the like. Nucleic acid can be linked to a solidsupport without a linker or with a linker (e.g., S. S. Wong, “Chemistryof Protein Conjugation and Cross-Linking,” CRC Press (1991), and G. T.Hermanson, “Bioconjugate Techniques,” Academic Press (1995). A homo orhetero-biofunctional linker reagent, can be selected, and examples oflinkers include without limitation N-succinimidyl(4-iodoacetyl)aminobenzoate (STAB), dimaleimide, dithio-bis-nitrobenzoic acid (DTNB),N-succinimidyl-S-acetyl-thioacetate (SATA),N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP), succinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC),6-hydrazinonicotimide (HYNIC), 3-amino-(2-nitrophenyl)propionic acid andthe like.

Nucleic acid can be synthesized using standard methods and equipment,such as the ABI®3900 High Throughput DNA Synthesizer and EXPEDITE®8909Nucleic Acid Synthesizer, both of which are available from AppliedBiosystems (Foster City, Calif.). Analogs and derivatives are describedin U.S. Pat. Nos. 4,469,863; 5,536,821; 5,541,306; 5,637,683; 5,637,684;5,700,922; 5,717,083; 5,719,262; 5,739,308; 5,773,601; 5,886,165;5,929,226; 5,977,296; 6,140,482; WO 00/56746; WO 01/14398, and relatedpublications. Methods for synthesizing nucleic acids containing suchanalogs or derivatives are disclosed, for example, in the patentpublications cited above and in U.S. Pat. Nos. 5,614,622; 5,739,314;5,955,599; 5,962,674; 6,117,992; in WO 00/75372 and in relatedpublications. In certain embodiments, analog nucleic acids includeinosines, abasic sites, locked nucleic acids, minor groove binders,duplex stabilizers (e.g., acridine, spermidine) and/or other meltingtemperature modifiers (e.g., target nucleic acid, solid phase nucleicacid, and/or primer nucleic acid may comprise an analog).

The density of solid phase-bound primer molecules per solid support unit(e.g., one bead or one sample location of a chip) can be selected. Amaximum density can be selected that allows for hybridization of samplenucleic acid from the plant cultivar to solid phase-bound primers. Incertain embodiments, solid phase-bound primer density per solid supportunit (e.g., nucleic acid molecules per bead) is about 5 nucleic acids toabout 10,000 nucleic acids per solid support. The density of the solidphase-bound primer per unit solid support in some embodiments can beabout 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85,90, 95, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000,4000, 5000, 6000, 7000, 8000, 9000 or 10000 nucleic acids per solidsupport. In certain embodiments the density of the solid phase-boundprimer per unit solid support is about 1 to 1 (e.g., one molecule ofsolid phase nucleic acid to one bead).

In certain embodiments, the solid supports can include any number ofprimer species useful for carrying out the analysis methods providedherein. Solid supports having primers attached or otherwise associatedthereto can be provided in any convenient form for contacting a samplenucleic acid from a plant cultivar, such as solid or liquid form, forexample. In certain embodiments, a solid support can be provided in aliquid form optionally containing one or more other components, whichinclude without limitation one or more buffers or salts. Solid supportsof a collection can be provided in one container or can be distributedacross multiple containers.

Solid supports can be provided in an array in certain embodiments, orinstructions can be provided to arrange solid supports in an array on asubstrate. The term “array” as used herein can refer to an arrangementof sample locations (for nucleic acid samples from plant cultivars) on asingle two-dimensional solid support, or an arrangement of solidsupports across a two-dimensional surface. An array can be of anyconvenient general shape (e.g., circular, oval, square, rectangular). Anarray can be referred to as an “X by Y array” for square or rectangulararrays, where the array includes X number of sample locations or solidsupports in one dimension and Y number of sample locations or solidsupports in a perpendicular dimension. An array can be symmetrical(e.g., a 16 by 16 array) or non-symmetrical (e.g., an 8 by 16 array). Anarray may include any convenient number of sample locations or solidsupports in any suitable arrangement. For example, X or Y independentlycan be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 in some embodiments.

An array can contain one solid support species or multiple solid supportspecies from a collection. The array can be arranged on any substratesuitable for sequence analysis or manufacture processes describedherein. Examples of substrates include without limitation flatsubstrates, filter substrates, wafer substrates, etched substrates,substrates having multiple wells or pits (e.g., microliter (about 1microliter, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75,80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200,300, 400, 500, 600, 700, 800, 900 and up to about 999 microlitervolume), nanoliter (1 nanoliter, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50,55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160,170, 180, 190, 200, 300, 400, 500, 600, 700, 800, 900 and up to about999 nanoliter volume), picoliter (1 picoliter, 5, 10, 15, 20, 25, 30,35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130,140, 150, 160, 170, 180, 190, 200, 300, 400, 500, 600, 700, 800, 900 andup to about 999 picoliter volume) wells or pits; wells having filterbottoms), substrates having one or more channels, substrates having oneor more electrodes, chips and the like, and combinations thereof. Wellsor pits of multiple well and pit substrates can contain one or moresolid support units (e.g., each unit being a single bead or particle).Substrates can include a suitable material for conducting sequenceanalysis or nucleic acid manufacture processes described herein,including without limitation, fiber (e.g., fiber filters), glass (e.g.,glass surfaces, fiber optic surfaces), metal (e.g., steel, gold, silver,aluminum, silicon and copper; metal coating), plastic (e.g.,polyethylene, polypropylene, polyamide, polyvinylidenedifluoride),silicon and the like. In certain embodiments, the array can be amicroarray or a nanoarray. A “nanoarray,” often is an array in whichsolid support units are separated by about 0.1 nanometers to about 10micrometers, for example from about 1 nanometer to about 1 micrometer(e.g. about 0.1 nanometers, 0.5, 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60,70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900 nanometers, 1micrometer, 2, 3, 4, 5, 6, 7, 8, 9, and up to about 10 micrometers). A“microarray” is an array in which solid support units are separated bymore than 1 micrometer. The density of solid support units on arraysoften is at least 100/cm², and can be 100/cm² to about 10,000/cm²,100/cm² to about 1,000/cm² or about 150, 200, 300, 400, 500, 600, 700,800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000 or 10000solid support units/cm².

Applications/Uses

The methods provided herein can additionally provide an outcomeindicative of one or more characteristics of a plant cultivar that maybe infected by a pathogen, including, but not limited to:

-   -   In an in-grow application setting, in a molecular lab        application setting, or as part of a kit of pathogen        identification markers.    -   Identifying more or less active variants of the pathogen genome        (e.g., HpLVd, AMV, BCTV or other pathogens or combinations        thereof, e.g., in multiplexed settings) for transgenic        experiments including CRISPR-cas9, Cre-Lox, and other genetic        modification applications to inhibit, silence, or interfere with        a more active variant or a less active variant.    -   Used in a cDNA microassay screening tool to identify presence        and/or amount of pathogen RNA present in a given Cannabis        cultivar.    -   Relating the amount of pathogen in a cell to the presentation or        absence of symptoms in infected plants.    -   Relating the genotype of pathogen in a cell to the presentation        or absence of symptoms in infected plants.    -   Relating a given pathogen (e.g., HpLVd, AMV, BCTV) genotype in        the panel to determining the performance, yield, and growth        characteristics of a given Cannabis cultivar.    -   Use of the markers (primers and/or the resulting amplicons) to        verify if clean stock treatments have removed or mutated the        pathogen genome from a given plant.    -   Identifying the mutant pathogen genome (e.g., HpLVd, AMV, BCTV)        to identify detrimental SNPs within the pathogen genome that        inhibit the viroid from affecting the host plants phenotype.    -   Use of the markers (primers and/or the resulting amplicons) to        identify plant genotypes that are resistant to certain variants        of the pathogen genome.

EXAMPLES

The examples set forth below illustrate certain embodiments and do notlimit the technology.

Example 1: Examples of Protocols for: (1) Determining the Presence,Absence and/or Amount of a Pathogen in a Plant Cultivar; (2) Determiningthe Genotype of the Pathogen RNA Isolation

Total RNA was isolated from fresh Cannabis leaf tissue samples using theDirect-zol RNA isolation kit and Zymo Research (Irvine, Calif.)Quick-RNA Plant Miniprep Kit with DNAase Digestion using manufacturerinstructions. Purified RNA was prepared for quantification using theQuantiFluor HS-ssRNA System (Promega, Madison, Wis.) and quantifiedusing a Quantus Fluorometer (Promega, Madison, Wis.), as permanufacturer's instructions. Quantified RNA was diluted to 5 ng/uL finalworking concentration and used as normalized input into either a firststrand cDNA synthesis reaction or one-step reverse transcriptasereal-time qPCR reactions.

cDNA Synthesis

Quantified RNA was used as input for cDNA synthesis using theSuperScript™ IV First-Strand Synthesis System (Thermo Fisher Scientific,Waltham, Mass.). cDNA synthesis reactions were prepared as follows: (1μL 50 μM Oligo d(T)20 primer, 1 μL of 10 mM dNTP mix (10 mM each), 8 μLTemplate RNA (10 pg-5 μg total RNA or 10 μg-500 ng mRNA) up to 3 μLDEPC-treated water for 13 μL final volume). After mixing and brieflycentrifuging, the RNA-primer mix reactions were heated at 65° C. for 5minutes, and then incubated at 0° C. for 2 minutes on a veritithermocycler.

Following annealing, the plate was pierced using a plate piercer and 7μL Reverse transcriptase (RT) reaction mix was added to each reactionfor a 20 μL final volume for cDNA synthesis. The RT reaction mix wasprepared as follows: 4 μL of 5×SSIV Buffer, 1 μL of 100 mM DTT, 1 μL ofRibonuclease Inhibitor, 1 μL of SuperScript™ IV Reverse Transcriptase(200 U/μL)). The plate was sealed and briefly centrifuged and loaded ona veriti thermocycler for cDNA synthesis using the following protocol:incubate the combined reaction mixture at 50-55° C. for 10 minutes,inactivate the reaction by incubating it at 80° C. for 10 minutes, andhold at 4° C. The products of cDNA synthesis were prepared forquantification using the QuantiFluor HS-dsDNA System (Promega, Madison,Wis.) and quantified using a Quantus Fluorometer (Promega, Madison,Wis.), as per manufacturer's instructions. Quantitated cDNA was dilutedto 2 ng/uL final working concentration and used as normalized input intoeither an end point PCR reaction or a Taqman real-time qPCR reaction.

Endpoint PCR with Gel Analysis 2.5 μL of normalized cDNA was used asinput into 22.5 μL of PCR master mix prepared per reaction as follows:12.5 μL 2× Promega Colorless GoTaq (Promega, Madison, Wis.), 0.1 μL of100 μM Primer Mixes, and 9.5 μL Nuclease free Water (Ambion, Austin,Tex.). The reactions were subjected to the following thermocyclerprotocol: 1 cycle at 95° C. for 10 mins; 35 cycles at 95° C. for 40 sec,60° C. for 2 mins, 72° C. for 2 mins; 1 cycle at 72° C. for 5 mins; 4°C. hold. End-point PCR reactions were analyzed by diluting 1:2 innuclease-free water and 20 μl was loaded into each well of E-Gel™ EXAgarose Gels, 2%, 20 gels and ran for 10 minutes on 1-2% gel settingsfor the E-gel system.

Reverse Transcriptase Quantitative Polymerase Chain Reaction (RT-qPCR)

RT-qPCR analysis was performed in 10 μL reactions on a LIGHTCYCLER 480qPCR (Roche Applied Systems, Germany) using the following protocol: 50°C. for 15 minutes hold, 95° C. for 2 minutes hold, followed by 40 cyclesof: 95° C. for 15 seconds, 60° C. for 30 seconds. Each reactioncontained: 2.5 μL of 5 ng/μL of the normalized RNA template used asinput, 7.5 μL of SUPERSCRIPT III PLATINUM One-step RT-qPCR Master Mix(prepared per reaction as follows: 5 μL One step RT-qPCR Master Mix(Thermo Fisher Scientific, Waltham, Mass.), 0.3 μL 10 μM primer, 0.25 μL10 μM probe, 13.6 μL H₂O, 0.25 μL TAQ). qPCR data was analyzed using theLIGHTCYCLER 480 software AbsQuant/2ndDerivative Max algorithm forcalculating Cp values.

Quantitative Real-Time PCR TAQMAN Analysis

qPCR analysis was performed in 10 μL reactions on a LIGHTCYCLER 480 qPCR(Roche Applied Systems, Germany) using the following protocol: 1pre-incubation cycle (95° C. for 20 secs), 45 amplification cycles (95°C. for 1 second, 60° C. for 20 seconds, 72° C. for 20 seconds) with asingle acquisition mode setting for each cycle at 60° C. annealing,followed by a final cooling cycle (40° C. for 30 seconds). Each reactioncontained: 2.5 μL of 2 ng/μL of the normalized cDNA template used asinput, 7.5 μL of TAQMAN Master Mix (prepared per reaction as follows: 5uL of FASTTQ Advanced Reaction Mix (Applied Biosciences, Beverly Hills,Calif.), 0.3 μL 10 μM primer, 0.25 μL 10 μM probe, 13.6 μL H₂O, 0.25 μLTAQ). qPCR data was analyzed using the LIGHTCYCLER 480 softwareAbsQuant/2ndDerivative Max algorithm for calculating Cp values.

High Resolution Melt (HRM) Analysis

HRM analysis was performed in 10 μL reactions on a LIGHTCYCLER 480 qPCR(Roche Applied Systems, Germany) using the following protocol: 1pre-incubation cycle (95° C. for 10 minutes), 45 amplification cycles(95° C. for 10 seconds, 60° C. for 15 seconds, 72° C. for 10 seconds), 1cycle of HRM (95° C. for 1 minute, 40° C. for 1 minute, 65° C. for 1second) and heat to 95° C. with 25 continuous acquisitions per degree(C.) followed by a final cooling cycle (40° C. for 10 seconds). Eachreaction contained: 2.5 μL of 2 ng/μL of the diluted pre-amplifiedtemplate, 7.5 μL of HRM Master Mix (prepared per reaction as follows: 5μL 2× High Resolution Melting Master Mix containing HRM dye (RocheApplied Systems, Germany), 0.6 μL of 4 μM Primer Mix, 0.8 μL of 25 mMMgCl₂, 1.125 μL of nuclease-free water). High Resolution Melting datawas analyzed using the LIGHTCYCLER 480 Melt Genotyping software.Fluorescence intensity as a function of temperature for each sample alsowas analyzed using R software custom scripts to determine statisticalvariation of melt curves.

Example 2: Methodologies to Identify Plants Affected by the Hops LatentViroid (HpLVd) and Classify the Genotype of the Viroid

This Example describes technology useful for identifying plants (e.g.,Cannabis plants) infected with HpLVd and, in certain instances,classifying the genotype of the viroid. A variety of moleculartechnologies may be used depending on the application desired.Applications include, for example, lab-based molecular diagnostics andin-field/cultivation facility diagnostics that can target of variety ofgenotypically different HpLVd genomes. Furthermore, this technology maybe useful within the process of clean-stock micropropagation and tissueculture, where heat treatment is a common method to remove the viroid.Heat treatment can mutate the HpLVd genome in certain regions, which canrender the viroid undetectable using existing primer designs. Theprimers provided in the Example overcome this by targeting conservedregions within thermomutants of HpLVd.

Components of the technology described in this Example includepolymerase chain reaction (PCR) primers, loop mediated isothermalamplification (LAMP) primers, RT-PCR primers, probes, and reversecomplements thereof. Primers and probes generally are about 15-30nucleotide-long sequences that are complementary to various loci of theHpLVd genome with purposely mismatched bases to loci in the Cannabisgenome CS10 Cannabis genome; GEN BANK assembly accession:GCA_900626175.1; REFSEQ assembly accession: GCF_900626175.1) to preventfalse positive results. Primer sequences provided in Table 1 below allowfor the identification of plants that contain the HpLVd RNA, and, incertain instances, classification of the genotype of the viroid throughvarious molecular technologies.

Amplification Primers and Amplification Products

One application of the amplification primers provided herein is agel-electrophoresis endpoint assay. Any combination of forward andreverse primers shown in Table 1 may be used in conjunction with an RNAlibrary or a cDNA library, and a corresponding size band (shown in Table2) in a gel from a combination of primers may be observed. In additionto using the primers as described below, the primers also can be used onwhole exome libraries, HpLVd specific libraries, and total RNA targetedcDNA libraries, as well as gene-specific cDNA synthesis as the firststep after RNA extraction to create only HpLVd cDNA without any hostplant cDNA being produced. All primer sets disclosed herein may be usedwithin a gene-specific cDNA synthesis protocol to amplify a region ofthe HpLVd genome that could be identified through a gel sizeidentification endpoint assay, or a high resolution melting (HRM)genotype endpoint assay, but only certain primers will work forgene-specific cDNA synthesis for a quantitative polymerase chainreaction (qPCR) endpoint. The amplicon lengths of each gene-specificcDNA target for each primer combination are shown in Table 2.

TABLE 1 Amplification primers Primer Sequence SEQ ID (type) (5′ to 3′)NO Length Start Stop A-fwd CTACGTGACTTAC 2 25 13 37 (tm- CTGTATGGTGGCspecific) A-rev CGCACGAACTGGC 3 18 106 89 (tm- GCTCG resistant) B-fwdGGGGAAACCTAC 4 19 60 78 (tm- TCGAGCG resistant) B-rev CTTCAGGTCGCC 5 19119 101 (tm- GCGCACG resistant) C-fwd GGAAACCTACTC 6 22 62 83 (tm-GAGCGAGGCG resistant) C-rev GTGAAGAAGGAG 7 20 171 152 (tm- CCGTTCCAspecific) D-rev CGGGTAGTTTCC 8 19 196 178 (tm- AACTCCG resistant) D-fwdCGAGGCGGAGAT 9 19 77 95 (tm- CGAGCGC resistant) E-rev CCGGGTAGTTTC 10 20197 178 (tm- CAACTCCG resistant) E-fwd GAGATCGAGCGC 11 19 84 102 (tm-CAGTTCG resistant) F-rev ACCGGGTAGTTT 12 21 198 178 (tm- CCAACTCCGresistant) F-fwd AGATCGAGCGC 13 18 85 102 (tm- CAGTTCG resistant) G-revAGAGTTGTATT 14 26 210 185 (tm- CACCGGGTAGT specific) TTCC H-revGCACTTTTTAT 15 23 252 230 (tm- GTGAACTTCT specific) GC

Several regions of the HpLVd genome were targeted for primer bindingregions with the intent that certain regions of the genome would be moreindicative of symptomatic plants than others. Certain mRNA transcriptsfrom cannabis and hops can be complimentary to the HpLVd genome, and theprimers were designed, in part, to genotype different regions of theHpLVd genome and find regions that can be complementary to cannabistranscripts and may cause a phenotypic change in the plant as a resultof the infection.

Certain primers were designed to primarily target sites that areresistant to thermomutation, and may be referred to asthermomutant-resistant (tm-resistant) primers. Other primers (e.g.,complementary to sequences towards the 3′ and 5′ ends of the HpLVdgenome, where thermomutants are possible) were designed asvariant-specific primers, and may be referred to asthermomutant-specific (tm-specific) primers. Using both types ofprimers, most of the HpLVd genome may be genotyped to identify SNPs inthe genome that can cause symptoms in given cultivars.

Primers that bind to a site of variation (e.g., A-fwd, C-rev, G-rev, andH-rev) are considered thermomutant-specific primers, and are specific toa certain variant of HpLVd. Such primer targeting allows foramplification only virulent/symptomatic versions of the viroid, whileavoiding non-symptomatic variants that were mutated during heat-shocktreatment and may no longer affect the phenotype. Includingthermo-mutant specific primers in the assays described herein allows forselection of more or less virulent/infectious/symptom-causing variantsby targeting regions of thermomutation. In this Example, A-fwdhybridizes to a region containing potential thermomutant SNPs atnucleotide positions 26-30, 33, and 35 of SEQ ID NO:1. C-rev hybridizesto a region containing potential thermomutant SNPs at positions 157,162, 168, and 169 of SEQ ID NO:1. G-rev hybridizes to a regioncontaining potential thermomutant SNP at position 210 of SEQ ID NO:1.H-rev hybridizes to a region containing potential thermomutant SNPs atpositions 247 and 248 of SEQ ID NO:1.

In certain instances, thermomutant-specific primers may be indicative ofthe presence or absence of HpLVd (e.g., in non-heat treated plants), andin certain instances, thermomutant-specific primers fail to detect thepresence of HpLVd (e.g., in heat-treated plants containing one or morethermomutations in the primer binding region). In certain instances,thermomutant-specific primers are useful for genotyping the entiregenome of HpLVd, and performing a secondary test if positive todetermine if the plant has the specific variants being targeted usingthose primers (e.g., variants present in the amplicons). In one modifiedapplication, primers with the longest amplicons (e.g., A-fwd, G-rev,H-rev) may be used by allowing for non-specific binding by modifying thePCR protocol to have a greater annealing temperature (+5 degrees C. fromprotocol) which would allow these primers to overcome the few mismatchesthat may be present in a thermomutant. Additionally, certain knownmutant sites could be targeted using modified versions ofthermomutant-specific primers (e.g., modified versions of thethermomutant-specific primers listed in Table 1) by replacing one ormore nucleotides at the mismatched sites with one or more nonstandard ordegenerate nucleotides to allow for a wider range of amplification ofthe HpLVd genome variants. For example, one or more nonstandard ordegenerate nucleotides may be incorporated in A-fwd that replace one ormore nucleotides that correspond to nucleotide positions 26-30, 33,and/or 35 of SEQ ID NO:1. One or more nonstandard or degeneratenucleotides may be incorporated in C-rev that replace one or morenucleotides that correspond to nucleotide positions 157, 162, 168,and/or 169 of SEQ ID NO:1. A nonstandard or degenerate nucleotide may beincorporated in G-rev that replace the nucleotide that corresponds tonucleotide position 210 of SEQ ID NO:1. One or more nonstandard ordegenerate nucleotides may be incorporated in H-rev that replace one ormore nucleotides that correspond to nucleotide positions 247 and/or 248of SEQ ID NO:1.

Primers that are designed to avoid binding to sites of variation (e.g.,A-rev, B-fwd, B-rev, D-rev, D-fwd, E-rev, E-fwd, F-rev, and F-fwd) areconsidered thermomutant-resistant primers. Amplification products fromsuch primers can be indicative of HpLVd infection, regardless of whetheror not the plant was mutated under heat treatments. Such primers weredesigned to include thermomutant positions within the amplicon and notwithin the primed regions.

In certain applications, plants may be genotyped for variants presentwithin an amplicon by amplification using thermomutant-resistant primersfollowed by a high resolution melt (HRM) assay or nucleotide sequencing.Additionally, plants may be genotyped for variants present within aprimer binding site by amplification using thermomutant-specificprimers, which provide a presence/absence answer to whether or not thatvariant is present.

Using multiple primers targeting multiple regions of the HpLVd genome inthe methods described herein provides a robust verification that theviroid is present or absent, minimizing false-positive andfalse-negative rates. Additionally, the use of multiple primerstargeting multiple regions allows for an identification of genotypesthat correspond to symptomatic plants.

TABLE 2 Amplification products (bp) Arev Brev Crev Drev Erev Frev GrevHrev Afwd 93 106 158 183 184 185 197 239 Bfwd 46 59 111 136 137 138 150192 Cfwd 44 57 109 134 135 136 148 190 Dfwd 29 42 94 119 120 121 133 175Efwd 22 35 87 112 113 114 126 168 Ffwd 21 34 86 111 112 113 125 167

Specificity of HpLVd in the Order Rosidae

To determine the specificity of our primers, homology comparisons wereperformed between the HpLVd genome and other plants in the orderRosidae. Using Blastn, a word size of 7nt was searched for homologybetween the 256 bp of HpLVd with both the whole genome shotgun contigsof 625 databases of species and 369 databases of the transcriptomeshotgun assembly databases included in the order Rosidae. The analysisshowed that the HpLVd genome is not present within the genome ortranscriptome of any other species of the order Rosidae, suggesting thatthe primers are specific to the HpLVd genome and will not amplify anyoff-target species of plant. Furthermore, in order to confirm theseresults, the primer combination (B-fwd with F-rev) was checked usingNCBI's primer designer software that uses Primer3, to ensure bothgenomic and transcriptomic specificity in the order Rosidae, which wasobserved within a single-target amplification of only the HpLVd genomewith no off-target RNA or DNA amplifications. Thus, the primers providedherein were confirmed as specific to HpLVd and will not amplify anyspecies of the order Rosidae.

High Resolution Melt (HRM) Assay

Another method of using primers provided herein is a high resolutionmelt (HRM) endpoint assay. This type of assay allows the user togenetically classify a variant of the HpLVd (e.g., a variant that isaffecting a given cultivar). The primers provided herein were designedso that the number of different primer combinations maximizes thelikelihood of capturing nucleic acid differences. Such primercombinations may be useful for detecting (1) symptomatic vs asymptomaticHpLVd variants, (2) triggers that induce a switch from asymptomatic tosymptomatic life cycle, (3) HpLVd variants that spread more easily, and(4) HpLVd variants that plants have gained resistance against. Primerset combinations shown with an asterisk (*) in Table 3 can be usedwithin an HRM endpoint assay, on a cDNA or an RNA template undermanufacturer's instructions (with the exception of certain thermocyclerprograms described herein).

TABLE 3 Arev Brev Crev Drev Erev Frev Grev Hrev Afwd * * * * * * * *Bfwd * * * * * * Cfwd * * * * * * Dfwd * * * * * * Efwd * * * * * *Ffwd * * * * * *

High Resolution Melt (HRM) Analysis

HRM analysis was performed in 10 μL reactions on a LIGHTCYCLER 480 qPCR(Roche Applied Systems) using the following protocol: 1 pre-incubationcycle (95° C. for 10 minutes), 45 amplification cycles (95° C. for 10seconds, 60° C. for 15 seconds, 72° C. for 10 seconds), 1 cycle of HRM(95° C. for 1 minute, 40° C. for 1 minute, 65° C. for 1 second) and heatto 95° C. with 25 continuous acquisitions per degree (C.) followed by afinal cooling cycle (40° C. for 10 seconds). Each reaction contained:2.5 μL of 2 ng/μL of the diluted pre-amplified template, 7.5 μL of HRMMaster Mix (prepared per reaction as follows: 5 μL 2× High ResolutionMelting Master Mix containing HRM dye (Roche Applied Systems), 0.6 μL of4 μM Primer Mix, 0.8 μL of 25 mM MgCl₂, 1.125 μL of nuclease freewater). High Resolution Melting data was analyzed using the LIGHTCYCLER480 Melt Genotyping software. Fluorescence intensity as a function oftemperature for each sample also was analyzed using R software customscripts to determine statistical variation of melt curves.

A melt curve genotyping analysis was performed with the following primerpairs: A-A, A-B, and A-C, and the results are shown in FIG. 8. For thisassay, each condition was carried out in a duplex reaction on the RocheLIGHTCYCLER 480 real time instrument. Gel CZ1 was used as a positivecontrol and no template was used as a negative control. The followingknown positive samples: BS2.1, Gel CZ4, and Gel CZ3 were analyzed forvarying melting profiles of A-A, A-B, and A-C amplicons using the RocheLIGHTCYCLER 480 melt curve genotyping analysis algorithm. No differencesin melt curve profiles were observed for the test samples and each testsample showed similar fluorescence values and melt curve temperature,indicating all the test samples had the same genotype. No amplificationor fluorescence was observed in the no template control.

Quantitative Polymerase Chain Reaction (qPCR)

Another intended use of the primers provided herein is use incombination with the qPCR probes designated in Table 5. The combinationsof primers and probes that identify the viroid are shown in Table 4.These combinations can be used on a cDNA template or an RNA templatethat is extracted from the cultivar for testing.

TABLE 4 Arev Brev Crev Drev Erev Frev Grev Hrev Afwd probe 1 probe 1probe 1 probe 1 probe 1 probe 1 probe 2 probe 2 probe 2 probe 2 probe 2probe 2 probe 3 probe 3 probe 3 probe 3 probe 3 probe 2 probe 3 probe 4probe 4 probe 4 probe 4 probe 4 probe 4 probe 4 probe 5 probe 5 probe 5probe 5 probe 5 Bfwd probe 1 probe 1 probe 1 probe 1 probe 1 probe 1probe 2 probe 2 probe 2 probe 2 probe 2 probe 2 probe 3 probe 3 probe 3probe 3 probe 3 probe 2 probe 3 probe 4 probe 4 probe 4 probe 4 probe 4probe 4 probe 4 probe 5 probe 5 probe 5 probe 5 probe 5 Cfwd probe 1probe 1 probe 1 probe 1 probe 1 probe 1 probe 3 probe 3 probe 3 probe 3probe 3 probe 3 probe 5 probe 5 probe 5 probe 5 probe 5 Dfwd probe 1probe 1 probe 1 probe 1 probe 1 probe 1 probe 3 probe 3 probe 3 probe 3probe 3 probe 3 probe 5 probe 5 probe 5 probe 5 probe 5 Efwd probe 3probe 3 probe 3 probe 3 probe 3 probe 3 probe 5 probe 5 probe 5 probe 5probe 5 Ffwd probe 3 probe 3 probe 3 probe 3 probe 3 probe 3 probe 5probe 5 probe 5 probe 5 probe 5

TABLE 5 SEQ ID Probe Sequence NO Start Stop Probe 1 TCGTGCGCGGCGACCT 16100 115 Probe 2 CGGAGATCGAGCGCCAGTT 17 81 100 Probe 3 TGCGCGGCGACCTGAAGT18 103 120 Probe 4 AGGCGGAGATCGAGCGCCA 19 79 97 Probe 5TCCTGCGTGGAACGGCTCC 20 143 163

Example qPCR protocols performed with the primers and probes providedherein are described below.

Reverse Transcriptase Quantitative Polymerase Chain Reaction (RT-qPCR)

RT-qPCR analysis was performed in 10 μL reactions on a LIGHTCYCLER 480qPCR (Roche Applied Systems) using the following protocol: 50° C. for 15minutes hold, 95° C. for 2 minutes hold, followed by 40 cycles of: 95°C. for 15 seconds, 60° C. for 30 seconds). Each reaction contained: 2.5μL of 5 ng/μL of the normalized RNA template used as input, 7.5 μL ofSUPERSCRIPT III PLATINUM One-step RT-qPCR Master Mix (prepared perreaction as follows: 5 μL One step RT-qPCR Master Mix (ThermoFisher),0.3 μL 10 μM primer, 0.10 μL-0.25 μL 10 μM probe, 13.6 μL H₂O, 0.25 μLTAQ). qPCR data was analyzed using the LIGHTCYCLER 480 softwareAbsQuant/2ndDerivative Max algorithm for calculating Cp values.

An optimization of general assay components for a hops latent viroidRT-qPCR method was performed, and the results are shown in FIG. 1. Eightconditions of varying RT-qPCR master mix compositions with differentconcentrations of primers, probe, water, and Taq for the primer pair A-Gand probe p1 were tested. Each reaction tube contained the volumesdescribed in FIG. 1 for each reagent component comprising a total volumeof 19 μL. Three samples were tested in this experiment, 1) a knownpositive-GelCZ1, 2) a known negative-GG #4 5.1, and 3) no template(water). To each reaction, 1 μL of 5 ng/μL RNA or Water was used astemplate input for a final reaction volume of 20 μL. For this assay,each condition was carried out in a single reaction on the RocheLIGHTCYCLER 480 real time instrument. Conditions labeled in FIG. 1 as 1,2, 4, 6, and 7 yielded detectable signals of fluorescence crossing athreshold value while conditions labeled in FIG. 1 as 3, 5, and 8yielded no detectable signal as is called by the Roche LIGHTCYCLERanalysis software 2nd derivative max analysis algorithm. Condition 7 wasselected to perform subsequent downstream testing as it preserved mastermix stock as well as had little to no background/late cycleamplification as was observed in the known negative sample forconditions 1, 2, and 4.

Further analysis of primer/probe combinations was performed for primerpairs A-D (FIG. 2), A-E (FIG. 2), A-F (FIG. 2), A-G (FIG. 3) B-D (FIG.3), B-E (FIG. 3), B-F (FIG. 4), B-G (FIG. 4) each tested with probesp1-p5 with optimized reaction condition 7 (shown in FIG. 1). For thispreliminary assay, each condition was carried out in a single reactionon the Roche LIGHTCYCLER 480 real time instrument. A reaction mix wasprepared for each test sample with each reaction containing RT-qPCRcomponents from condition 7 of: 5 μL Master mix, 0.3 μL 10 μM primerpair, 0.1 μL 10 μM probe, 13.6 μL nuclease free water, and 0.25 μLpolymerase enzyme for 19 μL total reaction mix. Three samples weretested in this experiment, 1) a known positive-Gel CZ1, 2) a knownnegative-GG #4 5.1, and 3) no template (water). To each reaction, 1 μLof 5 ng/μL RNA or water was used as template input for a final reactionvolume of 20 μL. In each assay, a positive fluorescent signal wasdetected and called by the Roche LIGHTCYCLER analysis software 2ndderivative max analysis algorithm for the Gel CZ1 positive sample and nosignal was observed in the GG #4 5.1 negative sample or no templatecontrol.

A further analysis of the primer pair A-G with probe p1 and primer pairF-D with probe p3 was performed on known positive and negative testsamples, and the results are shown in FIG. 5. The reactions wereprepared as described above for the experiments shown in FIGS. 2-4. Inthe HpLVd A-G; p1 test, a robust FAM fluorescent signal was observed aspositive for the following known positive samples: Gel CZ1, BS2.1, GelCZ4, Gel CZ3, and Gel CZ2. Background/late cycle amplification wasobserved for known negative samples: BBM #4 5.1. No signal was detectedin the GSC 5.3 sample, the GG #4 5.1 sample, or the no template control.In the HpLVd A-F; p3 test, a robust FAM fluorescent signal was observedas positive for the following known positive samples: Gel CZ1, BS2.1,Gel CZ4, Gel CZ3, and Gel CZ2. Background/late cycle amplification wasobserved for known negative samples: BBM #4 5.1 and GSC 5.3. No signalwas detected GG #4 5.1 sample or the no template control. To minimizebackground amplification, DNAase I digestion of RNA template materialand/or AMPErase reaction UNG pretreatment may be performed.

An analysis of unknown test samples was performed with the primer pairA-G with probe p1 and primer pair B-G with probe p5 using Gel CZ1 as apositive control and no template as a negative control, and the resultsare shown in FIG. 6. For this assay, each condition was prepared asdescribed above with duplicate replicates. Data acquisition and analysiswas performed on the Applied Biosystems QUANTSTUDIO 5 real timeinstrument and cloud software. An amplification status flag was appliedby software, and if amplification was observed for FAM channel,background or not, it is called an Amp. If no amplification is observed,the Amp status was observed as no Amp. No signal was measured on VICchannel. Positive and negative results were called based on an end pointfluorescence threshold. In the HpLVd A-G; p1 test, a robust FAMfluorescent signal was observed in the positive control replicates forGel CZ1 as well as the following samples: PP1 and SQR2. Background/latecycle amplification with end point fluorescence below threshold wasobserved for the following samples: GG #4 5.3, RH5.2, RH5.3, SQR3. Nofluorescence was detected in the BS2.3 sample, Ven 4.2 sample, andVen4.3 sample or no template control. In the HpLVd B-G; p5 test, arobust FAM fluorescent signal was observed in the positive controlreplicates for Gel CZ1 as well as the following samples: PP1 and SQR2.No background/late cycle amplification with end point fluorescence belowthreshold was observed. No fluorescence was detected in the followingsamples: GG #4 5.3, RH5.2, RH5.3, SQR3, BS2.3, Ven 4.2, Ven4.3, or notemplate control.

An analysis of genomic DNA and test RNA/cDNA samples was performed withthe primer pair A-G with probe p1 and primer pair B-G with probe p5using Gel CZ1 as a positive control and no template as a negativecontrol, and the results are shown in FIG. 7. This experiment wasperformed to demonstrate that no off target amplification occurred ingenomic DNA template. For this assay, each condition was prepared asdescribed above with duplicate replicates. 1 μL of 5 ng/μL RNA/cDNA/gDNAor water was used as template input. Data acquisition and analysis wasperformed on the Applied Biosystems QUANTSTUDIO 5 real time instrumentand cloud software. An amplification status flag was applied bysoftware, and if amplification was observed for FAM channel, backgroundor not, it is called an Amp. If no amplification is observed, the Ampstatus was observed as no Amp. No signal was measured on VIC channel.Positive and negative results were called based on an end pointfluorescence threshold. In both HpLVd A-G; p1 test and B-G; p5 test, arobust FAM fluorescent signal was observed in the positive controlreplicates for Gel CZ1 as well as the following samples: Gel 5.1 cDNA,Gel 5.1 Fresh FTA Card RNA, and Gel 5.1 Fresh Leaf RNA. Nobackground/late cycle amplification with end point fluorescence belowthreshold was observed. No fluorescence or amplification was detected inthe following samples: 9.5 Old FTA Card RNA, BK13419 gDNA, BK48007 gDNA,Crag 107-8 Old FTA Card RNA, Crag 108-4 Old FTA Card RNA, Durban PoisongDNA, G17 gDNA, G3 gDNA, OCBG gDNA, or no template control.

Quantitative Real-Time PCR TAQMAN Analysis

A TAQMAN protocol is another method in which the primers describedherein may be used. TAQMAN starts from a cDNA library instead ofextracted RNA (e.g., used as input for RT-qPCR). qPCR analysis wasperformed in 10 μL reactions on a LIGHTCYCLER 480 qPCR (Roche AppliedSystems) using the following protocol: 1 pre-incubation cycle (95° C.for 20 secs), 45 amplification cycles (95° C. for 1 second, 60° C. for20 seconds, 72° C. for 20 seconds) with a single acquisition modesetting for each cycle at 60° C. annealing, followed by a final coolingcycle (40° C. for 30 seconds). Each reaction contained: 2.5 μL of 2ng/μL of the normalized cDNA template used as input, 7.5 μL of TAQMANMaster Mix (prepared per reaction as follows: 5 μL of FASTTQ AdvancedReaction Mix (Applied Biosciences), 0.3 μL 10 μM primer, 0.10 μL-0.25 μL10 μM probe, 13.6 μL H₂O, 0.25 μL TAQ). qPCR data was analyzed using theLIGHTCYCLER 480 software AbsQuant/2ndDerivative Max algorithm forcalculating Cp values.

Loop Mediated Isothermal Amplification (LAMP)

Loop mediated isothermal amplification (LAMP) primers were designed foruse as a presence-absence test within a grow or lab environment. Theseprimers provide the user a readily detectable color change if the viroidis present, providing a time-saving and cost-effective solution toidentify infected plants within a grow. The primer sets designated inTables 6 to 9 are used in this methodology under standard reactionconditions following manufacturer instructions for a traditional LAMPassay. Each of the primer sets below were designed for use as a singleset. Accordingly four unique assays were created. The FIP (forward innerprimer), BIP (backward inner primer), F3 (forward outer primer) and B3(backward outer primer) primers may be generated with any loop (LB orLF) primers. The BIP and FIP are combinations of the B1c and B2, and F1c& F2 respectively, and may be linked by a polyT stretch that replacesthe “-” in the tables below. In a typical LAMP assay, BIP, FIP, F3, B3,and any loop primers (if they exist) are combined with a master mixsolution (provided by Eiken, Lucigen or a comparable LAMP master mixprovider) and an extracted cDNA solution. If the target sequence ispresent in cDNA synthesized from extracted RNA, upon incubation, a colorchange of the solution is observed due to a successful amplification ofthe target. An example LAMP primer scheme is provided in FIG. 9 and anexample LAMP assay is provided in FIG. 10.

The LAMP primers were designed as thermomutant-resistant primers;however, due to their longer size and the number of primers in each set,certain primers bind to thermomutant SNP sites. Generally, these primerswere designed such that the known SNP sites are located in the middle ofthe primed region to allow for amplification of mutant viroids. Foursets of LAMP primers were designed to provide the most robust assay thatwould be most thermomutant stable.

TABLE 6  LAMP Set 1 (dimer (minimum) dG = −2.18) SEQ la- 5′ 3′ 5′ 3′ Se-ID bel pos pos len Tm dG dG GCrate quence NO F3 39  56 18 59.55 −6.42−5.2 0.61 AGGGG 21 TCGA AGAG GGATC B3 208 225 18 60.31 −4.32 −4.27 0.56TAAGC 22 TCGG CGCTC AAGA FIP 39 CGAAG 23 CAACT TCAGG TCGCC G−CCC GGGGAAACCT ACTCG BIP 41 CTTCT 24 CCTTG TTCGC GTCCT GC−CC GGGTA GTTTC CAACT CCF2 57  74 18 60.75 −7.14 −4.76 0.67 CCCGG 25 GGAAA CCTA CTCG F1c 107 12721 65.74 −6.03 −7.71 0.62 CGAAG 26 CAACT TCAGG TCGCC G B2 179 197 1959.09 −7.12 −4.85 0.58 CCGGG 27 TAGTT TCCAA CTCC B1c 129 150 22 65.43−4.2 −6.1 0.59 CTTCT 28 CCTTG TTCGC GTCCT GC LB 158 178 21 65.01 −6.54−6.69 0.62 GGCTC 29 CTTCT TCACA CCAGC C

TABLE 7 LAMP Set 2 (dimer (minimum) dG = −2.18) SEQ la- 5′ 3′ 5′ 3′ Se-ID bel pos pos len Tm dG dG GCrate quence NO  F3 39  56 18 59.55 −6.42−5.2 0.61 AGGGC 30 TCGA AGAGG GATC B3 208 225 18 60.31 −4.32 −4.27 0.58TAAGC 31 TCGG CGCTC AAGA FIP 39 CGAAG 32 CAAC TTCAG GTCG CCG−C CCGGGGAAA CCTA CTCG BIP 42 CTTCT 33 CCTT GTTCG CGTC CTGC− ATCC ACCGG GTAGTTTC CAA F2 57  74 18 60.75 −7.14 −4.76 0.67 CCCGG 34 GGAA ACCTA CTCGF1c 107 127 21 65.74 −6.03 −7.71 0.62 CGAAG 35 CAAC TTCAG GTCG CCG B2183 202 20 60.61 −4.9 −4.53 0.5 ATCCA 36 CCGG GTAGT TTCC AA B1c 129 15022 65.43 −4.2 −6.1 0.59 CTTCT 37 CCTT GTTCG CGTC CTGC LB 158 178 2185.01 −6.54 −6.69 0.62 GGCTC 38 CTTC TTCAC ACCA GCC 

TABLE 8 LAMP Set 3 (dimer (minimum) dG = −2.18) SEQ la- 5′ 3′ 5′ 3′ IDbel pos pos len Tm dG dG GCrate Sequence NO F3 39 56 18 59.55 −6.42 −5.20.61 AGGGCTCG 39 AAGAGGGA TC B3 209 226 18 59.21 −4.09 −4.35 0.56TTAAGCTC 40 GGCGCTCA AG FIP 39 CGAAGCAA 41 CTTCAGGT CGCCG−CC CGGGGAAACCTACTCG BIP 42 CTTCTCCT 42 TGTTCGCG TCCTGC−A GTTGTATC CACCGGGT AGT F257 74 18 60.75 −7.14 −4.76 0.67 CCCGGGGA 43 AACCTACT CG F1c 107 127 2165.74 −6.03 −7.71 0.62 CGAAGCAA 44 CTTCAGGT CGCCG B2 189 208 20 59.69−4.55 −4.57 0.5 AGTTGTAT 45 CCACCGGG TAGT B1c 129 150 22 65.43 −4.2 −6.10.59 CTTCTCCT 46 TGTTCGCG TCCT GC LB 170 186 17 60.91 −5.56 −5 0.65ACACCAGC 47 CGGAGTTG G

TABLE 9 LAMP Set 4 (dimer (minimum) dG = −2.18) SEQ la- 5′ 3′ 5′ 3′ IDbel pos pos len Tm dG dG GCrate Sequence NO F3 39 56 18 59.55 −6.42 −5.20.61 AGGGCTCGA 48 AGAGGGATC B3 209 226 18 59.21 −4.09 −4.35 0.56TTAAGCTCG 49 GCGCTCAAG FIP 39 CGAAGCAAC 50 TTCAGGTCG CCG−CCCGG GGAAACCTACTCG BIP 41 CTTCTCCTT 51 GTTCGCGTC CTGC−CCGG GTAGTTTCC AACTCC F2 57 7418 60.75 −7.14 −4.76 0.67 CCCGGGGAA 52 ACCTACTCG F1c 107 127 21 65.74−6.03 −7.71 0.62 CGAAGCAAC 53 TTCAGGTCG CCG B2 179 197 19 59.09 −7.12−4.85 0.58 CCGGGTAGT 54 TTCCAACTC C B1c 129 150 22 65.43 −4.2 −6.1 0.59CTTCTCCTT 55 GTTCGCGTC CTGC LB 158 178 21 65.01 −6.54 −6.69 0.62GGCTCCTTC 56 TTCACACCA GCC

Reverse Complement Primers and Probes

The reverse complement of the primers provided in Table 1 and the probesprovided in Table 5 are provided in Table 10 and Table 11, respectively.

TABLE 10 SEQ Sequence ID Primer Name (5′->3′) Length NO A-fwd-RevCompGCCACCATACAGG 25 57 TAAGTCACGTAG A-rev-RevComp CGAGCGCCAGTTCGTGCG 18 58B-fwd-RevComp CGCTCGAGTAGGTTTCCCC 19 59 B-rev-RevCompCGTGCGCGGCGACCTGAAG 19 60 C-fwd-RevComp CGCCTCGCTCGAGTAGGTTTCC 22 61C-rev-RevComp TGGAACGGCTCCTTCTTCAC 20 62 D-rev-RevCompCGGAGTTGGAAACTACCCG 19 63 D-fwd-RevComp GCGCTCGATCTCCGCCTCG 19 64E-rev-RevComp CGGAGTTGGAAACTACCCGG 20 65 E-fwd-RevCompCGAACTGGCGCTCGATCTC 19 66 F-rev-RevComp CGGAGTTGGAAACTACCCGGT 21 67F-fwd-RevComp CGAACTGGCGCTCGATCT 18 68 G-rev-RevComp GGAAACTACCCGGTGA 2669 ATACAACTCT H-rev-RevComp GCAGAAGTTCACA 23 70 TAAAAAGTGC

TABLE 11 Sequence SEQ ID Probe (5′->3′) NO Probe 1-RevCompAGGTCGCCGCGCACGA 71 Probe 2-RevComp AACTGGCGCTCGATCTCCG 72Probe 3-RevComp ACTTCAGGTCGCCGCGCA 73 Probe 4-RevCompTGGCGCTCGATCTCCGCCT 74 Probe 5-RevComp GGAGCCGTTCCACGCAGGA 75

Applications

The technology described in this Example may be used in a number ofapplications, including, for example, in a cultivation facilityapplication setting, in a molecular lab application setting, and/or aspart of a kit of pathogen identification markers. Certain applicationsmay identify more or less active variants of the HpLVd genome fortransgenic experiments including CRISPR-cas9, Cre-Lox, and other geneticmodification applications to inhibit, silence, or interfere with themore or less active variant.

Certain applications may use this technology in a cDNA microassayscreening tool to identify presence and/or amount of viroid RNA presentin a given cannabis cultivar. Such applications may be useful for one ormore of 1) relating the amount of viroid in a cell to the presentationor absence of symptoms in infected plants, 2) relating the genotype ofviroid in a cell to the presentation or absence of symptoms in infectedplants, and 3) relating a given HpLVd genotype to determining theperformance, yield, and/or growth characteristics of a given cannabiscultivar.

Certain applications may use this technology to verify if in vitrotreatments have removed or mutated the HpLVd genome from a given plant.Such applications may be useful for identifying the mutant HpLVd genometo identify detrimental SNPs within the HpLVd genome that inhibit theviroid from affecting the host plants phenotype.

Certain applications may use this technology to identify plant genotypesthat are resistant to certain variants of the HpLVd genome. For example,LOX-1 variants that are non-complementary to the HpLVd genome may beidentified.

Example 3: Validation of a Multiplexed Method of Determining thePresence, Absence and/or Amount of More than One Pathogen in a PlantCultivar

Total RNA was collected from several Cannabis samples and a lyophilizedAMV-positive control sample using the kit from Zymo Research asdescribed in Example 1. The total RNA was carried through first strandcDNA synthesis.

To demonstrate broad qualitative sensitivity of high and low input, thefollowing samples were assayed by qPCR:

-   (1) a pool of cDNA from HpLVd-positive plant material normalized to    Ing and 0.1 ng and spiked with 1 ng and 0.1 ng AMV Positive Control    cDNA in 10 technical replicates,-   (2) a pool of cDNA from HpLVd and AMV-negative plant material; and-   (3) a no template control (NTC) of water

The samples were assayed using HPLVd, AMV, and 26S ribosomal RNA (rRNA)(internal positive control that is specific for cDNA from the plantgenome) in a multiplexed format. Samples assayed were tested across 3HPLVd primer pairs: HPLVd B-D (SEQ ID NOS: 4 and 8), HPLVd B-E (SEQ IDNOS: 4 and 10), and HPLVd B-F (SEQ ID NOS: 4 and 12) with all 5 HPLVdprobes p1-p5 (SEQ ID NOS:16-20) labeled with 6-FAM in a multiplex withthe AMV B-C primer pair (SEQ ID NOS: 82 and 84) with AMV probe B (SEQ IDNO:89) labeled with Cy5, and 1 internal positive control 26S rRNA primerpair (SEQ ID NOS: 107 and 108) with 26S rRNA probe p1 (SEQ ID NO:109)labeled with SUN (or VIC). cDNA, as described above, was used as inputinto the qPCR assay. The qPCR was formulated using Taqman Fast AdvancedMaster Mix (Thermo Fisher Scientific, Waltham, Mass.) and optimizedprimer/probe mix formulations. Cq mean and standard deviation values forHpLVd quantitation were calculated and observed to be relativelyconsistent across all primer and probe set multiplex combinations and inthe presence of various amounts of AMV cDNA spiking, as shown in thevalidation table in FIG. 11A. HPLVd detection of 1 ng, which is thestandard input for the assay, was consistently observed having Cq valuesbetween 20-21. The relative uniformity across high (1 ng cDNA) and low(0.1 ng) input Cq values for all HPLVd B-D, B-E, B-F primer setsdemonstrates reproducibility and broad sensitivity for detection of theHpLVd pathogen in HpLVd-positive plant samples, using HPLVd probes p1-5,in a multiplex assay in the presence of spiked amounts of cDNA from theAMV pathogen. FIG. 11B shows amplification plots using various primerpair/probe combinations (indicated at the top left) for detecting 26S(positive control; pale gray), HPLVd (medium gray) and AMV (dark gray).

Example 4: Multiplexed RT-qPCR for Determining the Presence, Absenceand/or Amount of the HpLVd, AMV and BCTV Pathogens in Cannabis Cultivars

In this analysis, four Cannabis cultivar plant samples from thegreenhouse were tested: two mother plants named CW14T520 M001 andCW14T520 M002, and two samplings from a symptomatic Abagail plant. TotalRNA was isolated as described in Example 2. For each reaction, Ing ofquantified and normalized RNA was used as input into an RT-qPCR one stepmultiplex reaction. The CW14T520 M001 and CW14T520 M002 mother plantsand the two samples from the Abagail symptomatic plant (Sample Reps 1and 2, see FIG. 12) were tested for the HpLVd pathogen using the B-FHPLVd primer pair (SEQ ID NOS: 4 and 12) with HPLVd probe p4 (SEQ IDNO:19) labeled with 6-FAM in a multiplex with the AMV pathogen B-Cprimer pair (SEQ ID NOS: 82 and 84) with AMV probe B (SEQ ID NO:89)labeled with Cy5, and 1 internal positive control 26S rRNA primer pair(SEQ ID NOS: 107 and 108) with 26S rRNA probe p1 (SEQ ID NO:109) labeledwith SUN (or VIC). The two mother plants, CW14T520 M001 and CW14T520M002 plants had an undetermined Cq value for AMV and HPLVd indicatingvalues below the threshold and therefore indicating the absence of thosepathogens in the mother plants. The two samples from the symptomaticAbagail plant tested negative for AMV, with an undetermined Cq value forAMV, and positive for HpLVd, with a Cq value above the threshold and astrong amplification curve observed (FIGS. 12A and 12B; in theamplification plots, 26S is pale gray, HPLVd or BCTV is medium gray; AMVis dark gray). All reactions in this multiplex tested positive for theinternal positive control 26S rRNA, with a Cq value crossing thethreshold and thereby indicating a successful RT-qPCR reaction.

The two samplings of the symptomatic Abagail plant were also tested forthe HpLVd pathogen using the B-F HPLVd primer pair (SEQ ID NOS: 4 and12) with HPLVd probe p4 (SEQ ID NO:19) labeled with 6-FAM in a multiplexwith the BCTV pathogen DRP_MP primer pair (SEQ ID NOS: 93 and 94) withBCTV Probe 1_DRP_MP (SEQ ID NO:95) labeled with Cy5, and 1 internalpositive control 26S rRNA primer pair (SEQ ID NOS: 107 and 108) with 26SrRNA probe p1 (SEQ ID NO:109) labeled with SUN (or VIC). The samplesfrom the symptomatic plant again tested positive for HpLVd, with similarCq values to those observed in the multiplex reaction described above.In addition, the samples from the symptomatic plant tested positive forBCTV, with a Cq value crossing the threshold and a strong amplificationcurve observed (FIGS. 12A and 12B; in the amplification plots, 26S ispale gray, HPLVd or BCTV is medium gray; AMV is dark gray). 1 ng and 5ng of a Gelato Cannabis sample that was positive for HpLVd was used as acontrol, along with a AMV positive control sample. Cq values were allobserved as expected, and no Cq values for any target was observed(below threshold) in the no template controls (NTC). These resultsdemonstrate selectivity for the individual pathogen targets insymptomatic and test plants from cultivation.

Example 5: Reproducibility of Multiplexed qPCR for Determining thePresence, Absence and/or Amount of the HpLVd, and BCTV Pathogens inPooled Leaf Samples from Cannabis Cultivars

In this analysis, ten plants were tested from a Cannabis cultivationfacility/greenhouse in Salinas, Calif. Total RNA was isolated asdescribed in Example 2. Five pooled leaf samples from ICC mother plantsand five pooled leaf samples from BSC mother plants were tested in atotal of ten test samples, each with 5 samples per pool (see FIGS. 13Aand 13B). For each reaction, 1 ng of quantified and normalized RNA wasused as input into an RT-qPCR one step multiplex reaction. The tenpooled samples were tested using the B-F HPLVd primer pair (SEQ ID NOS:4 and 12) with HPLVd probe p3 (SEQ ID NO:18) labeled with 6-FAM in amultiplex with the BCTV pathogen DRP_MP primer pair (SEQ ID NOS: 93 and94) with BCTV Probe 1_DRP_MP (SEQ ID NO:95) labeled with Cy5, and 1internal positive control 26S rRNA primer pair (SEQ ID NOS: 107 and 108)with 26S rRNA probe p1 (SEQ ID NO:109) labeled with SUN (or VIC). 4 outof 5 pools for each of the BSC and ICC samples had an undetermined Cqvalue for BCTV and HpLVd (below threshold), indicating the absence ofthose pathogens. One BSC pool (BSC-C) and one ICC pool (ICC-B) testednegative for BCTV, with an undetermined (below threshold) Cq value forBCTV, and positive for HpLVd, with a Cq value that was above thethreshold and a strong amplification curve observed (FIGS. 13A and 13B;in the amplification plots, 26S is pale gray, HPLVd is medium gray; BCTVis dark gray). All reactions in this multiplex tested positive for theinternal positive control 26S rRNA, with a Cq value crossing thethreshold and indicating a successful RT-qPCR reaction.

RNA from the symptomatic Abagail sample (1 ng, 0.1 ng, and 0.01 ngobtained by serial dilution) were used as positive controls and testedfor HpLVd using the using the B-F HPLVd primer pair (SEQ ID NOS: 4 and12) with HPLVd probe p4 (SEQ ID NO:19) labeled with 6-FAM in a multiplexwith the BCTV pathogen DRP_MP primer pair (SEQ ID NOS: 93 and 94) withBCTV Probe 1_DRP_MP (SEQ ID NO:95) labeled with Cy5, and 1 internalpositive control 26S rRNA primer pair (SEQ ID NOS: 107 and 108) with 26SrRNA probe p1 (SEQ ID NO:109) labeled with SUN (or VIC). The positivecontrol samples all tested positive for HpLVd, with similar Cq values tothose observed in the multiplex reaction described above. In addition,the positive control samples tested positive for the BCTV pathogen, witha Cq value crossing the threshold and a strong amplification curveobserved. The results demonstrate that the multiplexing method fordetermining the presence, absence and/or amounts of multiple samplesfrom plant cultivars can reliably be used to analyze multiple samplessimultaneously (e.g., amplified using different sets of primers, and/orfor detecting more than one pathogen).

Example 6: Sensitivity of Multiplexed RT-qPCR

In this analysis, two Cannabis plant samples were tested: onesymptomatic Abagail Hemp plant and one Cannabis RNA pool from HpLVdpositive plants at a concentration of 200 ng/uL that was combined at a1:1 v/v with the AMV positive control.

1 ng of quantified and normalized RNA from each sample was seriallydiluted in a ten-fold dilution series, down to 0.00001 ng, and used asinput into RT-qPCR one step multiplex reactions. The Abagail serialdilution standard curve was tested using the B-F HpLVd primer pair (SEQID NOS: 4 and 12) with HpLVd probe p3 (SEQ ID NO:18) labeled with 6-FAMin a multiplex with the BCTV pathogen DRP_MP primer pair (SEQ ID NOS: 93and 94) with BCTV Probe 1_DRP_MP (SEQ ID NO:95) labeled with Cy5, and 1internal positive control 26S rRNA primer pair (SEQ ID NOS: 107 and 108)with 26S rRNA probe p1 (SEQ ID NO:109) labeled with SUN (or VIC). TheAbagail sample showed strong amplification curves down to 100 fg forHPLVd and down to 10 pg for BCTV, with a series of Cq values thatcrossed the threshold up to the lowest levels of sensitivity for eachprimer pair under these conditions (FIG. 14A and FIG. 14B; in theamplification plots, 26S is pale gray, HPLVd is medium gray; AMV or BCTVis dark gray). These results indicate sensitivity of the HpLVd and BCTVprimers in a multiplex assay. All reactions in this multiplex testedpositive for the internal positive control 26S with a Ct value crossingthreshold indicating a successful RT-qPCR reaction down to the 100 fginput. No signal was observed below that input level. The 1:1 AMV spikedcannabis HpLVd+ pool dilution series were tested for HpLVd using the B-FHpLVd primer pair with the HpLVd probe p4 labeled with 6-FAM inmultiplex with AMV B-C primer pair AMV B-C with AMV probe B labeled withCy5, and 1 internal positive control 26S ribosomal RNA primer pair with26S probe p1 labeled with SUN. The AMV spiked Cannabis HpLVd+ pool hadstrong amplification curves down to 100 fg for HPpLVd and 10 fg for AMVwith a series of Ct values observed crossing threshold until lowestlevels of sensitivity for each primer pair under these conditions (FIG.14A and FIG. 14B; in the amplification plots, 26S is pale gray, HPLVd ismedium gray; AMV or BCTV is dark gray). These results indicate highsensitivity of the HpLVd and AMV primers in a multiplex assay. Allreactions in this multiplex tested positive for the internal positivecontrol 26S rRNA, with a Cq value crossing the threshold value, therebyindicating a successful RT-qPCR reaction down to the 100 fg amount thatwas input. No signal for either multiplex was observed in the notemplate control (NTC). FIG. 14C depicts standard curves for thepathogens in various samples as indicated at the top left of each curve.

Example 7: Robustness, Sensitivity, Specificity and Equivalency ofMultiplexed RT-qPCR and LAMP Assays

This example demonstrates the robustness, sensitivity, specificity andequivalency of Multiplexed RT-qPCR and LAMP Assays, and furtherdemonstrates that the LAMP colorimetric assay can serve as an accurate,simple, visual alternative to the RT-qPCR method for multiplexeddetection of pathogens in a plant.

In this analysis, total RNA was collected from several Cannabis samplesand an AMV lyophilized positive control using a commercial Plant QuickRNA kit (Zymo Research, Irvine, Calif.). Two sample pools of CannabisRNA were prepared: one from HPLVd, AMV and BCTV negative samples and theother pool prepared and formulated with HPLVd Positive Cannabis RNAsamples, HPLVd Positive and BCTV Positive Cannabis RNA samples and AMVpositive RNA samples. All positive and negative RNA pools were preparedat a final concentration of 1 ng/uL. A standard curve was alsoformulated to assess sensitivity, with serial 10-fold dilutions from 1ng/uL to 0.00001 ng/uL.

To demonstrate robust qualitative sensitivity and specificity fordetection of HPLVd, BCTV, and AMV in a RT-qPCR assay, both positive andnegative Cannabis RNA pools, an RNA standard curve, and a no templatecontrol (NTC) were used as input and assayed as duplicates. 1 uL ofinput was tested in HPLVd/AMV/26S and HPLVd/BCTV/26S RT-qPCR multiplexassays. A HPLVd/AMV/26S multiplex assay was prepared by formulating iTaqone step RT-qPCR Mastermix (Bio-Rad, Hercules, Calif.) with the B-FHPLVd primer pair and the HPLVd probe p4 labeled with 6-FAM in multiplexand HPLVd probe p2 labeled with ROX NHS, the AMV A-C primer pair withAMV probe B labeled with Cy5 and AMV probe A labeled with TAMRA NHS, and1 internal positive control 26S ribosomal RNA primer pair with 26S probep1 labeled with SUN. A second HPLVd/BCTV/26S multiplex was prepared byformulating iTaq one step RT-qPCR mastermix with the B-F HPLVd primerpair with the HPLVd probe p4 labeled with 6-FAM in multiplex and HPLVdprobe p2 labeled with ROX NHS, the BCTV DRP MP primer pair with BCTV DRPMP Probe 2 labeled with Cy5 and BCTV DRP MP Probe 1 labeled with TAMRANHS, and 1 internal positive control 26S ribosomal RNA primer pair with26S probe p1 labeled with SUN.

The results are shown in FIG. 15. FIG. 15 depicts the results for thenegative pools (no HPLVd, AMV or BCTV) on the left top and bottompanels, and the results for the positive pools (positive for HPLVd, AMV,BCTV) on the right top and bottom panels. In the HPLVd AMV 5 TargetMultiplex, both replicates for the 1 ng Cannabis RNA pool that was HPLVdand AMV negative showed a signal for the internal positive control 26SRNA having a Cq value that crossed the threshold, indicating asuccessful RT-qPCR reaction; no Cq values crossing the threshold wereobserved for HPLVd or AMV were observed (top left). In both technicalreplicates of the 1 ng Cannabis RNA pool that was HPLVd and AMVpositive, a signal for the internal positive control 26S was observedwith a Cq value crossing threshold, indicating a successful RT-qPCRreaction as well as Cq values for HPLVd p2 and p4 and AMV A and AMV Bwere observed indicating duplex target positive detection for HPLVd andAMV in the positive pool (top right). In the standard curve reaction,duplicate positive signals were observed for HPLVd p4 and p2 probes downto 100 fg, AMV A probe sensitivity down to 10 fg, AMV B probesensitivity down to 100 fg and 26S positive control sensitivity down to10 fg. No Cq values were obtained for any probe in the no templatecontrol.

In the HPLVd BCTV 5 Target Multiplex, both replicates for the 1 ngCannabis RNA pool that were HPLVd and BCTV Negative (negative pool)showed a signal for the internal positive control 26S with a Cq valuecrossing the threshold, indicating a successful RT-qPCR reaction; no Cqvalues crossing the threshold were obtained for HPLVd or BCTV (bottomleft). In both replicates of the 1 ng Cannabis RNA pool that were HPLVdand BCTV positive (positive pool), a signal for the internal positivecontrol 26S was observed, with a Cq value crossing threshold indicatinga successful RT-qPCR reaction. In addition, Cq values that crossed thethreshold were observed for HPLVd p2 and p4 probes and BCTV DRP MP Probe1 and Probe 2, indicating duplex target positive detection for HPLVd andBCTV in the positive pool (bottom right). In the standard curvereaction, duplicate positive signals were observed for HPLVd p4 and p2probes down to 100 fg, BCTV DRP MP Probe 1 and Probe 2 down to 1 pg, and26S sensitivity down to 10 fg. No Cq value observations were obtainedfor any probe in the no template control.

To evaluate the equivalency of the LAMP assay and the RT-qPCR assay formultiplexed detection of plant pathogens, crude RNA extract preparationand analysis of 24 different samples were carried out in duplicate for48 test reactions, along with a positive template control and notemplate control for RT-qPCR HPLVd detection. High throughput RT-qPCRMethod validation was carried out on crude extracts by preparingduplicate FTA Card sampling of leaf material, carrying through 96-wellplate preparation and extraction with a nucleic acid extraction buffer.Subsequently, crude extracts underwent one-step cDNA synthesis and preamplification using iTaq one-step Mastermix (Bio-Rad, Hercules, Calif.)and HPLVd B-F and 26S primers. Standard iTaq RT-PCR protocol conditionswere followed with 10 cycles of amplification.

Following the RT-PCR pre-amp protocol, pre-amp reactions were dilutedwith 100 uL of water and 5 uL was used as input into a qPCR reaction.The qPCR was formulated with Taqman Fast Advanced master mix (ThermoFisher, Fremond, Calif.) with HPLVd B-F Primers and 26S primers andprobes labeled with 6-FAM for HPLVd p4 and NHS Rox for HPLVd p2, and SUNfor 26S. Results of the test HPLVd positive and negatives samples wereevaluated as HPLVd positive or negative based on detection of a Cq valuefor HPLVd target probes that crossed the amplification curve Cqthreshold. Signals were observed in the HPLVd positive template control(26S) and with the HPLVd p4 and HPLVd p2 probes in both replicates,while no signal was observed in the no template control. Positive testsamples showed a Cq value with HPLVd p4, HPLVdp2 and 26S probes, whileNegative test samples only showed a Cq value for the positive control26S probe. The results are shown in FIG. 16.

Results obtained using the qPCR method (see above) were compared toresults in a subsequent analysis using the LAMP method and following theLAMP method evaluation for specificity and sensitivity. Total RNA from acommercial kit (Quick Plant RNA Kit, ZymoResearch, Irvine, Calif.) andcrude RNA extract (prepared as described previously) was used in theLAMP Method evaluation. 4 LAMP primer sets were initially tested with acouple of concentration levels of purified RNA, to gauge primer setsensitivity and performance. A positive LAMP reaction is observed when areaction tube changes from pink (seen as gray in grayscale, see FIG. 17)to yellow (seen as pale/transparent in grayscale, see FIG. 17) as targetamplicons accumulate. LAMP reactions with purified RNA as input wereprepared with NEB WarmStart Colorimetric LAMP Mastermix Mix (New EnglandBiolabs, Ipswich, Mass.) and HPLVd LAMP Primer Sets 1-4. The bestsensitivity and performance was observed with HPLVd LAMP Primer Sets 1and 2, with detection of HPLVd RNA down to 200 fg.

The HPLVd LAMP Primer Set 1 was carried through specificity andsensitivity validation of the high throughput method, and a qPCRequivalency study, using crude extracts. LAMP reactions carried out withcrude extracts first underwent one-step cDNA synthesis and preamplification using iTaq one-step mastermix (Bio-rad, Hercules, Calif.)and HPLVd LAMP Primer Set 1 B3 and F3 primers. Standard iTaq RT-PCRprotocol conditions were followed with 10 cycles of amplification.Following RT-PCR preamp protocol, Pre Amp reactions were diluted with100 uL of water and 1 uL was used as input into a LAMP reaction preparedwith NEB WarmStart Colorimetric LAMP Mastermix Mix (New England Biolabs,Ipswich, Mass.) and HPLVd LAMP Primer Set 1 B3, F3 FIP, BIP, and LB. Astandard curve was obtained using crude extract diluted in 10-foldseries from 10⁰ to 10−⁵ ng, along with a no template control (NTC).After 30 minutes at 65° C., a positive signal from the reaction at time0 (pink) turning to a yellow color could be observed all the way down to10−⁵ ng, while the NTC remained pink.

For evaluation of specificity a small test set of positive and negativesamples were prepared for crude extracts, which then underwent RT-PCRpreamp reactions followed by LAMP detection. At time 0 after addition oftemplate, the reactions remained pink. After 45 minutes at 65° C., apositive signal from the reaction turning to a yellow color could beobserved in the positive test samples and positive template control anda pink reaction color was observed in the negative test samples and inthe no template control. Observing a positive reaction color in thepositive samples and a negative reaction color in the negative samplesdemonstrates assay specificity.

To determine equivalency of the RT-qPCR and LAMP assays, follow upanalysis of HPLVd RT-qPCR method validation samples was carried outusing the LAMP method, and results between RT-qPCR and LAMP detectionmethods were compared. LAMP reactions were carried out with 24 samplestested in duplicate to yield 48 test crude extracts, as well as positivetemplate control and no template control samples. Samples firstunderwent one-step cDNA synthesis and pre-amplification using iTaqone-step Mastermix (Bio-Rad, Hercules, Calif.) and HPLVd LAMP Primer Set1 B3 and F3 primers. Standard RT-PCR protocol conditions were followedwith 10 cycles of amplification. Following RT-PCR pre-amp protocol,RT-PCR Pre Amp reactions were diluted with 100 uL of water and 1 uL wasused as input into a LAMP detection reaction prepared with NEB WarmStartColorimetric LAMP Mastermix Mix (New England Biolabs, Ipswich, Mass.)and HPLVd LAMP Primer Set 1 B3, F3 FIP, BIP, and LB. At time 0 afteraddition of template, samples were pink (the darker the gray shading,the deeper the pink color). Following 45 mins at 65° C., a positivesignal from reaction turning to a yellow color (depicted as pale samplesin FIG. 17; well numbers are indicated in the Table accompanying theFigure) could be observed in the positive test samples and positivetemplate control (PTC) and a pink reaction color was observed in thenegative test samples and in the no template control (NTC) (depicted asgray samples in FIG. 17; well numbers are indicated in the Tableaccompanying the Figure). Observing a color change in the positivesamples and no color change in the negative samples using this LAMPassays matches the RT-qPCR detection results and demonstratesequivalency of the two detection methods.

Example 8: Examples of Embodiments

The examples set forth below illustrate certain embodiments and do notlimit the technology.

-   A1. A method for analyzing nucleic acid from a plant sample,    comprising:    -   contacting nucleic acid of a plant sample with a plurality of        polynucleotide primer pairs under amplification conditions,        thereby preparing a mixture; and    -   analyzing nucleic acid of the mixture; wherein:-   the majority or all of the polynucleotide primer pairs hybridize to    subsequences of SEQ ID NO:1 if present in the nucleic acid of the    plant sample under the amplification conditions;-   the subsequences of SEQ ID NO:1 to which the majority or all of the    polynucleotide primers hybridize under the amplification conditions    contain no variant nucleotide position or one variant nucleotide    position; and-   each subsequence of SEQ ID NO:1 between the subsequences to which    the primer pairs hybridize contain two or more variant nucleotide    positions.-   A1.1 A method for preparing a nucleic acid mixture comprising:    -   contacting nucleic acid of a plant sample with a plurality of        polynucleotide primer pairs under amplification conditions,        thereby preparing a mixture, wherein:-   the majority or all of the polynucleotide primer pairs hybridize to    subsequences of SEQ ID NO:1 if present in the nucleic acid of the    plant sample under the amplification conditions;-   the subsequences of SEQ ID NO:1 to which the majority or all of the    polynucleotide primers hybridize under the amplification conditions    contain no variant nucleotide position or one variant nucleotide    position; and-   each subsequence of SEQ ID NO:1 between the subsequences to which    the primer pairs hybridize contain two or more variant nucleotide    positions.-   A1.2 The method of embodiment A1.1, comprising analyzing the nucleic    acid of the mixture.-   A2. A method for analyzing nucleic acid from a plant sample,    comprising:    -   contacting nucleic acid of a plant sample with one or more        polynucleotide primer pairs under amplification conditions,        thereby generating one or more amplification products; and    -   analyzing the amplification products; wherein:-   the majority or all of the one or more polynucleotide primer pairs    hybridize to subsequences of SEQ ID NO:1 if present in the nucleic    acid of the plant sample under the amplification conditions;-   the subsequences of SEQ ID NO:1 to which the majority or all of the    polynucleotide primers hybridize under the amplification conditions    contain no variant nucleotide position; and-   each subsequence of SEQ ID NO:1 between the subsequences to which    the one or more primer pairs hybridize contain one or more variant    nucleotide positions.-   A2.1 A method for generating nucleic acid amplification products    from a plant sample, comprising:    -   contacting nucleic acid of a plant sample with one or more        polynucleotide primer pairs under amplification conditions,        thereby generating one or more amplification products, wherein:-   the majority or all of the one or more polynucleotide primer pairs    hybridize to subsequences of SEQ ID NO:1 if present in the nucleic    acid of the plant sample under the amplification conditions;-   the subsequences of SEQ ID NO:1 to which the majority or all of the    polynucleotide primers hybridize under the amplification conditions    contain no variant nucleotide position; and-   each subsequence of SEQ ID NO:1 between the subsequences to which    the one or more primer pairs hybridize contain one or more variant    nucleotide positions.-   A2.2 The method of embodiment A2.1, comprising analyzing the    amplification products.-   A3. The method of embodiment A2, A2.1, or A2.2, comprising    contacting nucleic acid of a plant sample with a plurality of    polynucleotide primer pairs under amplification conditions.-   A4. The method of any one of embodiments A2 to A3, wherein each    subsequence of SEQ ID NO:1 between the subsequences to which the    primer pairs hybridize contain two or more variant nucleotide    positions.-   A5. The method of any one of embodiments A1 to A4, wherein each    subsequence of SEQ ID NO:1 between the subsequences to which the    primer pairs hybridize contain three or more variant nucleotide    positions.-   A6. The method of any one of embodiments A1 to A5, wherein each    subsequence of SEQ ID NO:1 between the subsequences to which the    primer pairs hybridize contain four or more variant nucleotide    positions.-   A7. The method of any one of embodiments A1, A.1, A1.2, and A3 to    A6, wherein the plurality of polynucleotide primer pairs comprises    two or more polynucleotide primer pairs.-   A8. The method of any one of embodiments A1, A1.1, A1.2, and A3 to    A7, wherein the plurality of polynucleotide primer pairs comprises    three or more polynucleotide primer pairs.-   A9. The method of any one of embodiments A1, A1.1, A1.2, and A3 to    A8, wherein the plurality of polynucleotide primer pairs comprises    four or more polynucleotide primer pairs.-   A10. The method of any one of embodiments A1, A1.1, A1.2, and A3 to    A9, wherein the plurality of polynucleotide primer pairs comprises    five or more polynucleotide primer pairs.-   All. The method of any one of embodiments A1, A1.1, A1.2, and A3 to    A10, wherein the plurality of polynucleotide primer pairs comprises    six or more polynucleotide primer pairs.-   A12. The method of any one of embodiments A1, A1.1, A1.2, and A3 to    A11, wherein the plurality of polynucleotide primer pairs comprises    seven or more polynucleotide primer pairs.-   A13. The method of any one of embodiments A1, A1.1, A1.2, and A3 to    A12, wherein the plurality of polynucleotide primer pairs comprises    eight or more polynucleotide primer pairs.-   A14. The method of any one of embodiments A1, A1.1, A1.2, and A3 to    A13, wherein the plurality of polynucleotide primer pairs comprises    nine or more polynucleotide primer pairs.-   A15. The method of any one of embodiments A1, A1.1, A1.2, and A3 to    A14, wherein the plurality of polynucleotide primer pairs comprises    ten or more polynucleotide primer pairs.-   A16. The method of any one of embodiments A1 to A15, wherein the    plant has been heat treated.-   A16.1 The method of any one of embodiments A1 to A15, wherein the    plant has not been heat treated.-   A16.1.1 The method of any one of embodiments A1 to A16.1, wherein    the plant is of the subclass Rosidae.-   A16.2 The method of any one of embodiments A1 to A16.1.1, wherein    the plant is a cannabis plant.-   A17. The method of embodiment A16.2, wherein each polynucleotide in    each primer pair comprises a sequence that is non-identical to any    subsequence, or complement thereof, in a cannabis genome.-   A18. The method of embodiment A17, wherein each polynucleotide in    each primer pair comprises a sequence that is non-identical to any    subsequence, or complement thereof, in a CS10 Cannabis genome.-   A19. The method of embodiment A18, wherein each polynucleotide in    each primer pair comprises a sequence comprising at least six    mismatches when compared to any subsequence, or complement thereof,    in the CS10 Cannabis genome.-   A20. The method of any one of embodiments A1 to A19, wherein each    polynucleotide in each primer pair comprises a sequence that is at    least about 90% identical to a subsequence, or complement thereof,    of SEQ ID NO:1.-   A21. The method of any one of embodiments A1 to A19, wherein each    polynucleotide in each primer pair comprises a sequence that is at    least about 95% identical to a subsequence, or complement thereof,    of SEQ ID NO:1.-   A22. The method of any one of embodiments A1 to A19, wherein each    polynucleotide in each primer pair comprises a sequence that is 100%    identical to a subsequence, or complement thereof, of SEQ ID NO:1.-   A23. The method of any one of embodiments A1 to A22, wherein each    primer pair comprises a forward primer and a reverse primer.-   A24. The method of embodiment A23, wherein each forward primer    hybridizes to a subsequence between nucleotide position 60 and    nucleotide position 102 of SEQ ID NO:1.-   A25. The method of embodiment A23 or A24, wherein each reverse    primer hybridizes to a subsequence between nucleotide position 89    and nucleotide position 119 of SEQ ID NO:1, or hybridizes to a    subsequence between nucleotide position 178 and nucleotide position    198 of SEQ ID NO:1.-   A26. The method of any one of embodiments A23 to A25, wherein one or    more forward primers independently are chosen from a polynucleotide    comprising a sequence that is at least about 90% identical to    GGGGAAACCTACTCGAGCG (SEQ ID NO:4), GGAAACCTACTCGAGCGAGGCG (SEQ ID    NO:6), CGAGGCGGAGATCGAGCGC (SEQ ID NO:9), GAGATCGAGCGCCAGTTCG (SEQ    ID NO:11), and AGATCGAGCGCCAGTTCG (SEQ ID NO:13).-   A27. The method of any one of embodiments A23 to A25, wherein one or    more forward primers independently are chosen from a polynucleotide    comprising a sequence that is at least about 95% identical to    GGGGAAACCTACTCGAGCG (SEQ ID NO:4), GGAAACCTACTCGAGCGAGGCG (SEQ ID    NO:6), CGAGGCGGAGATCGAGCGC (SEQ ID NO:9), GAGATCGAGCGCCAGTTCG (SEQ    ID NO:11), and AGATCGAGCGCCAGTTCG (SEQ ID NO:13).-   A28. The method of any one of embodiments A23 to A25, wherein one or    more forward primers independently are chosen from a polynucleotide    comprising a sequence that is 100% identical to GGGGAAACCTACTCGAGCG    (SEQ ID NO:4), GGAAACCTACTCGAGCGAGGCG (SEQ ID NO:6),    CGAGGCGGAGATCGAGCGC (SEQ ID NO:9), GAGATCGAGCGCCAGTTCG (SEQ ID    NO:11), and AGATCGAGCGCCAGTTCG (SEQ ID NO:13).-   A29. The method of any one of embodiments A23 to A28, wherein one or    more reverse primers independently are chosen from a polynucleotide    comprising a sequence that is at least about 90% identical to    CGCACGAACTGGCGCTCG (SEQ ID NO:3), CTTCAGGTCGCCGCGCACG (SEQ ID NO:5),    CGGGTAGTTTCCAACTCCG (SEQ ID NO:8), CCGGGTAGTTTCCAACTCCG (SEQ ID    NO:10), and ACCGGGTAGTTTCCAACTCCG (SEQ ID NO:12).-   A30. The method of any one of embodiments A23 to A28, wherein one or    more reverse primers independently are chosen from a polynucleotide    comprising a sequence that is at least about 95% identical to    CGCACGAACTGGCGCTCG (SEQ ID NO:3), CTTCAGGTCGCCGCGCACG (SEQ ID NO:5),    CGGGTAGTTTCCAACTCCG (SEQ ID NO:8), CCGGGTAGTTTCCAACTCCG (SEQ ID    NO:10), and ACCGGGTAGTTTCCAACTCCG (SEQ ID NO:12).-   A31. The method of any one of embodiments A23 to A28, wherein one or    more reverse primers independently are chosen from a polynucleotide    comprising a sequence that is 100% identical to CGCACGAACTGGCGCTCG    (SEQ ID NO:3), CTTCAGGTCGCCGCGCACG (SEQ ID NO:5),    CGGGTAGTTTCCAACTCCG (SEQ ID NO:8), CCGGGTAGTTTCCAACTCCG (SEQ ID    NO:10), and ACCGGGTAGTTTCCAACTCCG (SEQ ID NO:12).-   A32. The method of any one of embodiments A23 to A31, wherein the    plurality of polynucleotide primer pairs comprises a plurality of    forward primers and a plurality of reverse primers.-   A33. The method of embodiment A32, wherein the plurality of forward    primers comprises GGGGAAACCTACTCGAGCG (SEQ ID NO:4),    GGAAACCTACTCGAGCGAGGCG (SEQ ID NO:6), CGAGGCGGAGATCGAGCGC (SEQ ID    NO:9), GAGATCGAGCGCCAGTTCG (SEQ ID NO:11), and AGATCGAGCGCCAGTTCG    (SEQ ID NO:13); and the plurality of reverse primers comprises    CGCACGAACTGGCGCTCG (SEQ ID NO:3), CTTCAGGTCGCCGCGCACG (SEQ ID NO:5),    CGGGTAGTTTCCAACTCCG (SEQ ID NO:8), CCGGGTAGTTTCCAACTCCG (SEQ ID    NO:10), ACCGGGTAGTTTCCAACTCCG (SEQ ID NO:12), and    AGAGTTGTATTCACCGGGTAGTTTCC (SEQ ID NO:14).-   A34. The method of embodiment A32, wherein the plurality of forward    primers consists of GGGGAAACCTACTCGAGCG (SEQ ID NO:4),    GGAAACCTACTCGAGCGAGGCG (SEQ ID NO:6), CGAGGCGGAGATCGAGCGC (SEQ ID    NO:9), GAGATCGAGCGCCAGTTCG (SEQ ID NO:11), and AGATCGAGCGCCAGTTCG    (SEQ ID NO:13); and the plurality of reverse primers consists of    CGCACGAACTGGCGCTCG (SEQ ID NO:3), CTTCAGGTCGCCGCGCACG (SEQ ID NO:5),    CGGGTAGTTTCCAACTCCG (SEQ ID NO:8), CCGGGTAGTTTCCAACTCCG (SEQ ID    NO:10), and ACCGGGTAGTTTCCAACTCCG (SEQ ID NO:12).-   A35. The method of any one of embodiments A1 to A34, wherein the    analyzing comprises detecting the presence or absence of a hops    latent viroid in the plant.-   A36. The method of any one of embodiments A1 to A35, wherein the    analyzing comprises detecting one or more genetic variations in a    hops latent viroid.-   A37. The method of embodiment A36, wherein the analyzing comprises    detecting two or more genetic variations in a hops latent viroid.-   A38. The method of embodiment A36 or A37, wherein detecting the one    or more genetic variations in the hops latent viroid comprises    contacting the nucleic acid of the plant sample with one or more    further polynucleotide primers under amplification conditions,    wherein:    -   the majority or all of the further polynucleotide primers        hybridize to subsequences of SEQ ID NO:1 if present in the        nucleic acid of the plant sample under the amplification        conditions; and    -   the subsequences of SEQ ID NO:1 to which the majority or all of        the further polynucleotide primers hybridize under the        amplification conditions contain one or more variant nucleotide        positions.-   A39. The method of embodiment A38, wherein each further    polynucleotide primer comprises a sequence that is non-identical to    any subsequence, or complement thereof, in a cannabis genome.-   A40. The method of embodiment A39, wherein each further    polynucleotide primer comprises a sequence that is non-identical to    any subsequence, or complement thereof, in a CS10 Cannabis genome.-   A41. The method of embodiment A40, wherein each further    polynucleotide primer comprises a sequence comprising at least six    mismatches when compared to any subsequence, or complement thereof,    in the CS10 Cannabis genome.-   A42. The method of any one of embodiments A38 to A41, wherein each    further polynucleotide primer comprises a sequence that is at least    about 90% identical to a subsequence, or complement thereof, of SEQ    ID NO:1.-   A43. The method of any one of embodiments A38 to A41, wherein each    further polynucleotide primer comprises a sequence that is at least    about 95% identical to a subsequence, or complement thereof, of SEQ    ID NO:1.-   A44. The method of any one of embodiments A38 to A41, wherein each    further polynucleotide primer comprises a sequence that is 100%    identical to a subsequence, or complement thereof, of SEQ ID NO:1.-   A45. The method of any one of embodiments A38 to A44, wherein the    one or more further polynucleotide primers independently are chosen    from a polynucleotide comprising a sequence that is at least about    90% identical to CTACGTGACTTACCTGTATGGTGGC (SEQ ID NO:2),    GTGAAGAAGGAGCCGTTCCA (SEQ ID NO:7), AGAGTTGTATTCACCGGGTAGTTTCC (SEQ    ID NO:14), and GCACTTTTTATGTGAACTTCTGC (SEQ ID NO:15).-   A46. The method of any one of embodiments A38 to A44, wherein the    one or more further polynucleotide primers independently are chosen    from a polynucleotide comprising a sequence that is at least about    95% identical to CTACGTGACTTACCTGTATGGTGGC (SEQ ID NO:2),    GTGAAGAAGGAGCCGTTCCA (SEQ ID NO:7), AGAGTTGTATTCACCGGGTAGTTTCC (SEQ    ID NO:14), and GCACTTTTTATGTGAACTTCTGC (SEQ ID NO:15).-   A47. The method of any one of embodiments A38 to A44, wherein the    one or more further polynucleotide primers independently are chosen    from a polynucleotide comprising a sequence that is 100% identical    to CTACGTGACTTACCTGTATGGTGGC (SEQ ID NO:2), GTGAAGAAGGAGCCGTTCCA    (SEQ ID NO:7), AGAGTTGTATTCACCGGGTAGTTTCC (SEQ ID NO:14), and    GCACTTTTTATGTGAACTTCTGC (SEQ ID NO:15).-   A48. The method of any one of embodiments A38 to A44, wherein the    one or more further polynucleotide primers comprise    CTACGTGACTTACCTGTATGGTGGC (SEQ ID NO:2), GTGAAGAAGGAGCCGTTCCA (SEQ    ID NO:7), AGAGTTGTATTCACCGGGTAGTTTCC (SEQ ID NO:14), and    GCACTTTTTATGTGAACTTCTGC (SEQ ID NO:15).-   A49. The method of any one of embodiments A38 to A44, wherein the    one or more further polynucleotide primers consist of    CTACGTGACTTACCTGTATGGTGGC (SEQ ID NO:2), GTGAAGAAGGAGCCGTTCCA (SEQ    ID NO:7), AGAGTTGTATTCACCGGGTAGTTTCC (SEQ ID NO:14), and    GCACTTTTTATGTGAACTTCTGC (SEQ ID NO:15).-   A50. The method of any one of embodiments A36 to A49, wherein the    one or more genetic variations comprise one or more nucleotide    insertions.-   A51. The method of any one of embodiments A36 to A50, wherein the    one or more genetic variations comprise one or more nucleotide    deletions.-   A52. The method of embodiment A51, wherein the one or more    nucleotide deletions comprise a deletion at nucleotide position 225    of SEQ ID NO:1.-   A53. The method of any one of embodiments A36 to A52, wherein the    one or more genetic variations comprise one or more single    nucleotide variations.-   A54. The method of embodiment A53, wherein the one or more single    nucleotide variations comprise a variant nucleotide at one or more    of nucleotide position 7 of SEQ ID NO:1, nucleotide position 10 of    SEQ ID NO:1, nucleotide position 12 of SEQ ID NO:1, nucleotide    position 26 of SEQ ID NO:1, nucleotide position 27 of SEQ ID NO:1,    nucleotide position 28 of SEQ ID NO:1, nucleotide position 29 of SEQ    ID NO:1, nucleotide position 30 of SEQ ID NO:1, nucleotide position    33 of SEQ ID NO:1, nucleotide position 35 of SEQ ID NO:1, nucleotide    position 43 of SEQ ID NO:1, nucleotide position 59 of SEQ ID NO:1,    nucleotide position 121 of SEQ ID NO:1, nucleotide position 128 of    SEQ ID NO:1, nucleotide position 134 of SEQ ID NO:1, nucleotide    position 150 of SEQ ID NO:1, nucleotide position 157 of SEQ ID NO:1,    nucleotide position 162 of SEQ ID NO:1, nucleotide position 168 of    SEQ ID NO:1, nucleotide position 169 of SEQ ID NO:1, nucleotide    position 177 of SEQ ID NO:1, nucleotide position 200 of SEQ ID NO:1,    nucleotide position 225 of SEQ ID NO:1, nucleotide position 229 of    SEQ ID NO:1, nucleotide position 247 of SEQ ID NO:1, nucleotide    position 248 of SEQ ID NO:1, and nucleotide position 253 of SEQ ID    NO:1-   A55. The method of any one of embodiments A36 to A54, wherein the    analyzing comprises identifying a hops latent viroid trait according    to the one or more genetic variations.-   A56. The method of embodiment A36 to A54, wherein the analyzing    comprises detecting a genetic variation signature.-   A57. The method of embodiment A56, wherein the genetic variation    signature comprises genotypes determined at two or more variant    nucleotide positions.-   A58. The method of embodiment A56, wherein the genetic variation    signature comprises genotypes determined at three or more variant    nucleotide positions.-   A59. The method of embodiment A56, wherein the genetic variation    signature comprises genotypes determined at four or more variant    nucleotide positions.-   A60. The method of embodiment A56, wherein the genetic variation    signature comprises genotypes determined at five or more variant    nucleotide positions.-   A61. The method of embodiment A56, wherein the genetic variation    signature comprises genotypes determined at six or more variant    nucleotide positions.-   A62. The method of embodiment A56, wherein the genetic variation    signature comprises genotypes determined at seven or more variant    nucleotide positions.-   A63. The method of embodiment A56, wherein the genetic variation    signature comprises genotypes at determined eight or more variant    nucleotide positions.-   A64. The method of embodiment A56, wherein the genetic variation    signature comprises genotypes determined at nine or more variant    nucleotide positions.-   A65. The method of embodiment A56, wherein the genetic variation    signature comprises genotypes determined at ten or more variant    nucleotide positions.-   A66. The method of any one of embodiments A56 to A65, wherein the    analyzing comprises identifying a hops latent viroid trait according    to the genetic variation signature.-   A67. The method of any one of embodiments A1 to A66, wherein the    method further comprises contacting the nucleic acid of the plant    sample with one or more quantitative PCR probes under the    amplification conditions.-   A68. The method of embodiment A67, wherein the one or more    quantitative PCR probes are chosen from one or more of    TCGTGCGCGGCGACCT (SEQ ID NO:16), CGGAGATCGAGCGCCAGTT (SEQ ID NO:17),    TGCGCGGCGACCTGAAGT (SEQ ID NO:18), AGGCGGAGATCGAGCGCCA (SEQ ID    NO:19), and TCCTGCGTGGAACGGCTCC (SEQ ID NO:20).-   A69. The method of any one of embodiments A1 to A68, wherein the    method comprises contacting the nucleic acid of the plant sample    with a set of loop mediated isothermal amplification (LAMP) primers    under the amplification conditions.-   A70. The method of embodiment A69, wherein the LAMP primer set is    chosen from one or more of:    -   a) a primer set comprising the polynucleotides of SEQ ID NO:21        to SEQ ID NO:29,    -   b) a primer set comprising the polynucleotides of SEQ ID NO:30        to SEQ ID NO:38,    -   c) a primer set comprising the polynucleotides of SEQ ID NO:39        to SEQ ID NO:47, and    -   d) a primer set comprising the polynucleotides of SEQ ID NO:48        to SEQ ID NO:56.-   A71. The method of any one of embodiments A1, A1.1, and A3 to A70,    wherein the analyzing comprises performing a high resolution melting    (HRM) endpoint assay on the nucleic acid in the mixture.-   A72. The method of any one of embodiments A2 to A70, wherein the    analyzing comprises performing a high resolution melting (HRM)    endpoint assay on the amplification products.-   A73. The method of embodiment A72, wherein the analyzing comprises    detecting one or more genetic variations in a hops latent viroid    according to results obtained from the high resolution melting (HRM)    endpoint assay.-   A74. The method of embodiment A72, wherein the analyzing comprises    detecting two or more genetic variations in a hops latent viroid    according to results obtained from the high resolution melting (HRM)    endpoint assay.-   A75. The method of any one of embodiments A1 to A74, wherein the    subsequences of SEQ ID NO:1 to which the majority or all of the    polynucleotide primers hybridize under the amplification conditions    contain no thermomutant positions.-   A76. The method of embodiment A75, wherein the thermomutant    positions are chosen from one or more of nucleotide position 7 of    SEQ ID NO:1, nucleotide position 10 of SEQ ID NO:1, nucleotide    position 12 of SEQ ID NO:1, nucleotide position 26 of SEQ ID NO:1,    nucleotide position 27 of SEQ ID NO:1, nucleotide position 28 of SEQ    ID NO:1, nucleotide position 29 of SEQ ID NO:1, nucleotide position    30 of SEQ ID NO:1, nucleotide position 33 of SEQ ID NO:1, nucleotide    position 35 of SEQ ID NO:1, nucleotide position 43 of SEQ ID NO:1,    nucleotide position 59 of SEQ ID NO:1, nucleotide position 121 of    SEQ ID NO:1, nucleotide position 128 of SEQ ID NO:1, nucleotide    position 134 of SEQ ID NO:1, nucleotide position 150 of SEQ ID NO:1,    nucleotide position 157 of SEQ ID NO:1, nucleotide position 162 of    SEQ ID NO:1, nucleotide position 168 of SEQ ID NO:1, nucleotide    position 169 of SEQ ID NO:1, nucleotide position 177 of SEQ ID NO:1,    nucleotide position 200 of SEQ ID NO:1, nucleotide position 225 of    SEQ ID NO:1, nucleotide position 229 of SEQ ID NO:1, nucleotide    position 247 of SEQ ID NO:1, nucleotide position 248 of SEQ ID NO:1,    and nucleotide position 253 of SEQ ID NO:1.-   A77. A method for analyzing nucleic acid from a plant sample,    comprising:    -   a) contacting nucleic acid of a plant sample with a first set of        polynucleotide primers under amplification conditions, thereby        generating a first set of amplification products, wherein:        -   i) the majority or all of the primers in the first set of            polynucleotide primers hybridize to subsequences of SEQ ID            NO:1 if present in the nucleic acid of the plant sample            under the amplification conditions,        -   ii) the subsequences of SEQ ID NO:1 to which the majority or            all of the primers in the first set of polynucleotide            primers hybridize under the amplification conditions contain            no variant nucleotide position, and        -   iii) each subsequence of SEQ ID NO:1 between the            subsequences to which the primers in the first set of            polynucleotide primers hybridize contain one or more variant            nucleotide positions;    -   b) contacting the nucleic acid of the plant sample with a second        set of polynucleotide primers under the amplification        conditions, thereby generating a second set of amplification        products, wherein:        -   i) the majority or all of the primers in the second set of            polynucleotide primers hybridize to subsequences of SEQ ID            NO:1 if present in the nucleic acid of the plant sample            under the amplification conditions, and        -   ii) the subsequences of SEQ ID NO:1 to which the majority or            all of the primers in the second set of polynucleotide            primers hybridize under the amplification conditions contain            one or more variant nucleotide positions; and    -   c) analyzing the first and second sets of amplification        products.-   A78. A method for generating nucleic acid amplification products    from a plant sample, comprising:    -   a) contacting nucleic acid of a plant sample with a first set of        polynucleotide primers under amplification conditions, thereby        generating a first set of amplification products, wherein:        -   i) the majority or all of the primers in the first set of            polynucleotide primers hybridize to subsequences of SEQ ID            NO:1 if present in the nucleic acid of the plant sample            under the amplification conditions,        -   ii) the subsequences of SEQ ID NO:1 to which the majority or            all of the primers in the first set of polynucleotide            primers hybridize under the amplification conditions contain            no variant nucleotide position, and        -   iii) each subsequence of SEQ ID NO:1 between the            subsequences to which the primers in the first set of            polynucleotide primers hybridize contain one or more variant            nucleotide positions; and    -   b) contacting the nucleic acid of the plant sample with a second        set of polynucleotide primers under the amplification        conditions, thereby generating a second set of amplification        products, wherein:        -   i) the majority or all of the primers in the second set of            polynucleotide primers hybridize to subsequences of SEQ ID            NO:1 if present in the nucleic acid of the plant sample            under the amplification conditions, and        -   ii) the subsequences of SEQ ID NO:1 to which the majority or            all of the primers in the second set of polynucleotide            primers hybridize under the amplification conditions contain            one or more variant nucleotide positions.-   A79. The method of embodiment A78, comprising analyzing the first    and second sets of amplification products.-   A80. The method of any one of embodiments A77 to A79, comprising one    or more features of any one of embodiments A3 to A76.-   A81. The method of any one of embodiments A1 to A80 that is    performed on a FTA® card.-   B1. A composition comprising one or more polynucleotide primer pairs    wherein:    -   each polynucleotide of the one or more primer pairs is        identical, or substantially identical, to a subsequence of SEQ        ID NO:1, or complement thereof;    -   each subsequence of SEQ ID NO:1, or complement thereof, to which        each polynucleotide is identical, or substantially identical,        contains no variant nucleotide position; and    -   each target sequence of SEQ ID NO:1 between the subsequences, or        complements thereof, to which the polynucleotides of the one or        more primer pairs are identical, or substantially identical,        comprises one or more variant nucleotide positions.-   B2. Reserved.-   B3. The composition of embodiment B1, wherein each target sequence    of SEQ ID NO:1 comprises two or more variant nucleotide positions.-   B4. The composition of embodiment B1, wherein each target sequence    of SEQ ID NO:1 comprises three or more variant nucleotide positions.-   B5. The composition of embodiment B1, wherein each target sequence    of SEQ ID NO:1 comprises four or more variant nucleotide positions.-   B6. The composition of any one of embodiments B1 to B5, wherein the    one or more polynucleotide primer pairs comprise two or more    polynucleotide primer pairs.-   B7. The composition of any one of embodiments B1 to B5, wherein the    one or more polynucleotide primer pairs comprise three or more    polynucleotide primer pairs.-   B8. The composition of any one of embodiments B1 to B5, wherein the    one or more polynucleotide primer pairs comprise four or more    polynucleotide primer pairs.-   B9. The composition of any one of embodiments B1 to B5, wherein the    one or more polynucleotide primer pairs comprise five or more    polynucleotide primer pairs.-   B10. The composition of any one of embodiments B1 to B5, wherein the    one or more polynucleotide primer pairs comprise six or more    polynucleotide primer pairs.-   B11. The composition of any one of embodiments B1 to B5, wherein the    one or more polynucleotide primer pairs comprise seven or more    polynucleotide primer pairs.-   B12. The composition of any one of embodiments B1 to B5, wherein the    one or more polynucleotide primer pairs comprise eight or more    polynucleotide primer pairs.-   B13. The composition of any one of embodiments B1 to B5, wherein the    one or more polynucleotide primer pairs comprise nine or more    polynucleotide primer pairs.-   B14. The composition of any one of embodiments B1 to B5, wherein the    one or more polynucleotide primer pairs comprise ten or more    polynucleotide primer pairs.-   B15. The method of any one of embodiments B1 to B14, wherein each    polynucleotide in each primer pair comprises a sequence that is    non-identical to any subsequence, or complement thereof, in a    cannabis genome.-   B16. The composition of embodiment B15, wherein each polynucleotide    in each primer pair comprises a sequence that is non-identical to    any subsequence, or complement thereof, in a CS10 Cannabis genome.-   B17. The composition of embodiment B16, wherein each polynucleotide    in each primer pair comprises a sequence comprising at least six    mismatches when compared to any subsequence, or complement thereof,    in the CS10 Cannabis genome.-   B18. The composition of any one of embodiments B1 to B17, wherein    each polynucleotide in each primer pair comprises a sequence that is    at least about 90% identical to a subsequence, or complement    thereof, of SEQ ID NO:1.-   B19. The composition of any one of embodiments B1 to B17, wherein    each polynucleotide in each primer pair comprises a sequence that is    at least about 95% identical to a subsequence, or complement    thereof, of SEQ ID NO:1.-   B20. The composition of any one of embodiments B1 to B17, wherein    each polynucleotide in each primer pair comprises a sequence that is    100% identical to a subsequence, or complement thereof, of SEQ ID    NO:1.-   B21. The composition of any one of embodiments B1 to B20, wherein    each primer pair comprises a forward primer and a reverse primer.-   B22. The composition of embodiment B21, wherein each forward primer    is identical, or substantially identical, to a subsequence, or    complement thereof, between nucleotide position 60 and nucleotide    position 102 of SEQ ID NO:1.-   B23. The composition of embodiment B21 or B22, wherein each reverse    primer is identical, or substantially identical, to a subsequence,    or complement thereof, between nucleotide position 89 and nucleotide    position 119 of SEQ ID NO:1; or is identical, or substantially    identical, to a subsequence, or complement thereof, between    nucleotide position 178 and nucleotide position 198 of SEQ ID NO:1-   B24. The composition of any one of embodiments B21 to B23, wherein    one or more forward primers independently are chosen from a    polynucleotide comprising a sequence that is at least about 90%    identical to GGGGAAACCTACTCGAGCG (SEQ ID NO:4),    GGAAACCTACTCGAGCGAGGCG (SEQ ID NO:6), CGAGGCGGAGATCGAGCGC (SEQ ID    NO:9), GAGATCGAGCGCCAGTTCG (SEQ ID NO:11), and AGATCGAGCGCCAGTTCG    (SEQ ID NO:13).-   B25. The composition of any one of embodiments B21 to B23, wherein    one or more forward primers independently are chosen from a    polynucleotide comprising a sequence that is at least about 95%    identical to GGGGAAACCTACTCGAGCG (SEQ ID NO:4),    GGAAACCTACTCGAGCGAGGCG (SEQ ID NO:6), CGAGGCGGAGATCGAGCGC (SEQ ID    NO:9), GAGATCGAGCGCCAGTTCG (SEQ ID NO:11), and AGATCGAGCGCCAGTTCG    (SEQ ID NO:13).-   B26. The composition of any one of embodiments B21 to B23, wherein    one or more forward primers independently are chosen from a    polynucleotide comprising a sequence that is 100% identical to    GGGGAAACCTACTCGAGCG (SEQ ID NO:4), GGAAACCTACTCGAGCGAGGCG (SEQ ID    NO:6), CGAGGCGGAGATCGAGCGC (SEQ ID NO:9), GAGATCGAGCGCCAGTTCG (SEQ    ID NO:11), and AGATCGAGCGCCAGTTCG (SEQ ID NO:13).-   B27. The composition of any one of embodiments B21 to B26, wherein    one or more reverse primers independently are chosen from a    polynucleotide comprising a sequence that is at least about 90%    identical to CGCACGAACTGGCGCTCG (SEQ ID NO:3), CTTCAGGTCGCCGCGCACG    (SEQ ID NO:5), CGGGTAGTTTCCAACTCCG (SEQ ID NO:8),    CCGGGTAGTTTCCAACTCCG (SEQ ID NO:10), and ACCGGGTAGTTTCCAACTCCG (SEQ    ID NO:12).-   B28. The composition of any one of embodiments B21 to B26, wherein    one or more reverse primers independently are chosen from a    polynucleotide comprising a sequence that is at least about 95%    identical to CGCACGAACTGGCGCTCG (SEQ ID NO:3), CTTCAGGTCGCCGCGCACG    (SEQ ID NO:5), CGGGTAGTTTCCAACTCCG (SEQ ID NO:8),    CCGGGTAGTTTCCAACTCCG (SEQ ID NO:10), and ACCGGGTAGTTTCCAACTCCG (SEQ    ID NO:12).-   B29. The composition of any one of embodiments B21 to B26, wherein    one or more reverse primers independently are chosen from a    polynucleotide comprising a sequence that is 100% identical to    CGCACGAACTGGCGCTCG (SEQ ID NO:3), CTTCAGGTCGCCGCGCACG (SEQ ID NO:5),    CGGGTAGTTTCCAACTCCG (SEQ ID NO:8), CCGGGTAGTTTCCAACTCCG (SEQ ID    NO:10), and ACCGGGTAGTTTCCAACTCCG (SEQ ID NO:12).-   B30. The composition of any one of embodiments B21 to B29,    comprising a plurality of forward primers and a plurality of reverse    primers.-   B31. The composition of embodiment B30, wherein the plurality of    forward primers comprises GGGGAAACCTACTCGAGCG (SEQ ID NO:4),    GGAAACCTACTCGAGCGAGGCG (SEQ ID NO:6), CGAGGCGGAGATCGAGCGC (SEQ ID    NO:9), GAGATCGAGCGCCAGTTCG (SEQ ID NO:11), and AGATCGAGCGCCAGTTCG    (SEQ ID NO:13); and the plurality of reverse primers comprises    CGCACGAACTGGCGCTCG (SEQ ID NO:3), CTTCAGGTCGCCGCGCACG (SEQ ID NO:5),    CGGGTAGTTTCCAACTCCG (SEQ ID NO:8), CCGGGTAGTTTCCAACTCCG (SEQ ID    NO:10), and ACCGGGTAGTTTCCAACTCCG (SEQ ID NO:12).-   B32. The composition of embodiment B30, wherein the plurality of    forward primers consists of GGGGAAACCTACTCGAGCG (SEQ ID NO:4),    GGAAACCTACTCGAGCGAGGCG (SEQ ID NO:6), CGAGGCGGAGATCGAGCGC (SEQ ID    NO:9), GAGATCGAGCGCCAGTTCG (SEQ ID NO:11), and AGATCGAGCGCCAGTTCG    (SEQ ID NO:13); and the plurality of reverse primers consists of    CGCACGAACTGGCGCTCG (SEQ ID NO:3), CTTCAGGTCGCCGCGCACG (SEQ ID NO:5),    CGGGTAGTTTCCAACTCCG (SEQ ID NO:8), CCGGGTAGTTTCCAACTCCG (SEQ ID    NO:10), and ACCGGGTAGTTTCCAACTCCG (SEQ ID NO:12).-   B33. The composition of any one of embodiments B1 to B32, further    comprising one or more quantitative PCR probes.-   B34. The composition of embodiment B33, wherein the one or more    quantitative PCR probes are chosen from one or more of    TCGTGCGCGGCGACCT (SEQ ID NO:16), CGGAGATCGAGCGCCAGTT (SEQ ID NO:17),    TGCGCGGCGACCTGAAGT (SEQ ID NO:18), AGGCGGAGATCGAGCGCCA (SEQ ID    NO:19), and TCCTGCGTGGAACGGCTCC (SEQ ID NO:20).-   B35. The composition of any one of embodiments B1 to B34, comprising    a set of loop mediated isothermal amplification (LAMP) primers.-   B36. The composition of embodiment B35, wherein the LAMP primer set    is chosen from one or more of:    -   a) a primer set comprising the polynucleotides of SEQ ID NO:21        to SEQ ID NO:29,    -   b) a primer set comprising the polynucleotides of SEQ ID NO:30        to SEQ ID NO:38,    -   c) a primer set comprising the polynucleotides of SEQ ID NO:39        to SEQ ID NO:47, and    -   d) a primer set comprising the polynucleotides of SEQ ID NO:48        to SEQ ID NO:56.-   B37. The composition of any one of embodiments B1 to B36, wherein    each subsequence of SEQ ID NO:1, or complement thereof, to which    each polynucleotide is identical, or substantially identical,    contains no thermomutant positions.-   B38. The composition of embodiment B37, wherein the thermomutant    positions are chosen from one or more of nucleotide position 7 of    SEQ ID NO:1, nucleotide position 10 of SEQ ID NO:1, nucleotide    position 12 of SEQ ID NO:1, nucleotide position 26 of SEQ ID NO:1,    nucleotide position 27 of SEQ ID NO:1, nucleotide position 28 of SEQ    ID NO:1, nucleotide position 29 of SEQ ID NO:1, nucleotide position    30 of SEQ ID NO:1, nucleotide position 33 of SEQ ID NO:1, nucleotide    position 35 of SEQ ID NO:1, nucleotide position 43 of SEQ ID NO:1,    nucleotide position 59 of SEQ ID NO:1, nucleotide position 121 of    SEQ ID NO:1, nucleotide position 128 of SEQ ID NO:1, nucleotide    position 134 of SEQ ID NO:1, nucleotide position 150 of SEQ ID NO:1,    nucleotide position 157 of SEQ ID NO:1, nucleotide position 162 of    SEQ ID NO:1, nucleotide position 168 of SEQ ID NO:1, nucleotide    position 169 of SEQ ID NO:1, nucleotide position 177 of SEQ ID NO:1,    nucleotide position 200 of SEQ ID NO:1, nucleotide position 225 of    SEQ ID NO:1, nucleotide position 229 of SEQ ID NO:1, nucleotide    position 247 of SEQ ID NO:1, nucleotide position 248 of SEQ ID NO:1,    and nucleotide position 253 of SEQ ID NO:1.-   B39. The composition of any one of embodiments B1 to B38, comprising    one or more further polynucleotide primers wherein:    -   each polynucleotide of the one or more further polynucleotide        primers is identical, or substantially identical, to a        subsequence of SEQ ID NO:1, or complement thereof;    -   each subsequence of SEQ ID NO:1, or complement thereof, to which        each polynucleotide is identical, or substantially identical,        contains one or more variant nucleotide positions.-   B40. The composition of embodiment B39, wherein each further    polynucleotide primer comprises a sequence that is non-identical to    any subsequence, or complement thereof, in a cannabis genome.-   B41. The composition of embodiment B40, wherein each further    polynucleotide primer comprises a sequence that is non-identical to    any subsequence, or complement thereof, in a CS10 Cannabis genome.-   B42. The composition of embodiment B41, wherein each further    polynucleotide primer comprises a sequence comprising at least six    mismatches when compared to any subsequence, or complement thereof,    in the CS10 Cannabis genome.-   B43. The composition of any one of embodiments B39 to B42, wherein    each further polynucleotide primer comprises a sequence that is at    least about 90% identical to a subsequence, or complement thereof,    of SEQ ID NO:1.-   B44. The composition of any one of embodiments B39 to B42, wherein    each further polynucleotide primer comprises a sequence that is at    least about 95% identical to a subsequence, or complement thereof,    of SEQ ID NO:1.-   B45. The composition of any one of embodiments B39 to B42, wherein    each further polynucleotide primer comprises a sequence that is 100%    identical to a subsequence, or complement thereof, of SEQ ID NO:1.-   B46. The composition of any one of embodiments B39 to B45, wherein    the one or more further polynucleotide primers independently are    chosen from a polynucleotide comprising a sequence that is at least    about 90% identical to CTACGTGACTTACCTGTATGGTGGC (SEQ ID NO:2),    GTGAAGAAGGAGCCGTTCCA (SEQ ID NO:7), AGAGTTGTATTCACCGGGTAGTTTCC (SEQ    ID NO:14), and GCACTTTTTATGTGAACTTCTGC (SEQ ID NO:15).-   B47. The composition of any one of embodiments B39 to B45, wherein    the one or more further polynucleotide primers independently are    chosen from a polynucleotide comprising a sequence that is at least    about 95% identical to CTACGTGACTTACCTGTATGGTGGC (SEQ ID NO:2),    GTGAAGAAGGAGCCGTTCCA (SEQ ID NO:7), AGAGTTGTATTCACCGGGTAGTTTCC (SEQ    ID NO:14), and GCACTTTTTATGTGAACTTCTGC (SEQ ID NO:15).-   B48. The composition of any one of embodiments B39 to B45, wherein    the one or more further polynucleotide primers independently are    chosen from a polynucleotide comprising a sequence that is 100%    identical to CTACGTGACTTACCTGTATGGTGGC (SEQ ID NO:2),    GTGAAGAAGGAGCCGTTCCA (SEQ ID NO:7), AGAGTTGTATTCACCGGGTAGTTTCC (SEQ    ID NO:14), and GCACTTTTTATGTGAACTTCTGC (SEQ ID NO:15).-   B49. The composition of any one of embodiments B39 to B45, wherein    the one or more further polynucleotide primers comprise    CTACGTGACTTACCTGTATGGTGGC (SEQ ID NO:2), GTGAAGAAGGAGCCGTTCCA (SEQ    ID NO:7), AGAGTTGTATTCACCGGGTAGTTTCC (SEQ ID NO:14), and    GCACTTTTTATGTGAACTTCTGC (SEQ ID NO:15).-   B50. The composition of any one of embodiments B39 to B45, wherein    the one or more further polynucleotide primers consist of    CTACGTGACTTACCTGTATGGTGGC (SEQ ID NO:2), GTGAAGAAGGAGCCGTTCCA (SEQ    ID NO:7), AGAGTTGTATTCACCGGGTAGTTTCC (SEQ ID NO:14), and    GCACTTTTTATGTGAACTTCTGC (SEQ ID NO:15).-   B51. A composition comprising:    -   a) a first set of polynucleotide primers wherein:        -   i) each polynucleotide of the a first set of polynucleotide            primers is identical, or substantially identical, to a            subsequence of SEQ ID NO:1, or complement thereof,        -   ii) each subsequence of SEQ ID NO:1, or complement thereof,            to which each polynucleotide is identical, or substantially            identical, contains no variant nucleotide position, and        -   iii) each target sequence of SEQ ID NO:1 between the            subsequences, or complements thereof, to which the            polynucleotides of the first set of polynucleotide primers            are identical, or substantially identical, comprises one or            more variant nucleotide positions; and    -   b) a second set of polynucleotide primers wherein:        -   i) each polynucleotide of the second set of polynucleotide            primers is identical, or substantially identical, to a            subsequence of SEQ ID NO:1, or complement thereof, and        -   ii) each subsequence of SEQ ID NO:1, or complement thereof,            to which each polynucleotide is identical, or substantially            identical, contains one or more variant nucleotide            positions.-   B52. The composition of embodiment B51, comprising one or more    features from any one of embodiments B3 to B50.-   B53. A kit comprising the composition of any one of embodiments B1    to B52 and instructions for use.-   C1. A method for determining the presence, absence and/or amount of    a pathogen in a plant cultivar, comprising:    -   (a) obtaining a nucleic acid sample from the plant cultivar;    -   (b) contacting the nucleic acid sample with at least one        polynucleotide primer pair under amplification conditions and        amplifying the sample, thereby preparing an amplified nucleic        acid mixture, wherein, if the pathogen is present, the        polynucleotide primer pair is capable of specifically        hybridizing to and amplifying a subsequence of the nucleic acid        of the pathogen, or to a complement thereof, wherein the        subsequence of the nucleic acid of the pathogen, or the        complement thereof, is non-identical to any subsequence of the        nucleic acid of the plant genome, or to any complement thereof;        and    -   (c) determining the presence, absence and/or amount of at least        one amplicon that is 300 base pairs or less and is an        amplification product of the polynucleotide primer pair in the        amplified nucleic acid mixture of (b), thereby determining the        presence, absence and/or amount of a pathogen in the plant        cultivar.-   C1.1. A method of preparing a nucleic acid mixture from a plant    cultivar, comprising:    -   (b) obtaining a nucleic acid sample from the plant cultivar; and    -   (b) preparing an amplified nucleic acid mixture by contacting        the nucleic acid sample with at least one polynucleotide primer        pair under amplification conditions and amplifying the sample,        wherein, if the pathogen is present, the polynucleotide primer        pair is capable of specifically hybridizing to and amplifying a        subsequence of the nucleic acid of the pathogen, or to a        complement thereof, wherein the subsequence of the nucleic acid        of the pathogen, or the complement thereof, is non-identical to        any subsequence of the nucleic acid of the plant genome, or to        any complement thereof.-   C1.2. The method of embodiment C1.1, further comprising, determining    the presence, absence and/or amount of at least one amplicon that is    300 base pairs or less and is an amplification product of the    polynucleotide primer pair in the amplified nucleic acid mixture of    (b), thereby determining the presence, absence and/or amount of a    pathogen in the plant cultivar.-   C1.3. A method for determining the presence, absence and/or amount    of at least one pathogen in a plant cultivar, comprising:-   (a) obtaining a nucleic acid sample from the plant cultivar;-   (b) contacting the nucleic acid sample with more than one    polynucleotide primer pair under amplification conditions and    amplifying the sample, thereby preparing an amplified nucleic acid    mixture, wherein, if at least one pathogen is present, at least one    polynucleotide primer pair is capable of specifically hybridizing to    and amplifying a subsequence of the nucleic acid of the pathogen, or    to a complement thereof, wherein the subsequence of the nucleic acid    of the pathogen, or the complement thereof, is non-identical to any    subsequence of the nucleic acid of the plant genome, or to any    complement thereof; and-   (c) determining the presence, absence and/or amount of at least one    amplicon that is an amplification product of a polynucleotide primer    pair in the amplified nucleic acid mixture of (b), thereby    determining the presence, absence and/or amount of a pathogen in the    plant cultivar.-   C1.4. The method of embodiment C1.3, wherein:-   each of the polynucleotide primer pairs hybridizes to the nucleic    acid of the same pathogen;-   each polynucleotide primer pair hybridizes to a subsequence of the    nucleic acid of the pathogen that does not overlap with the    subsequences to which each of the other primer pairs hybridizes; and-   the presence, absence and/or amount of more than one amplicon of the    pathogen that is obtained in (b) is determined in (c).-   C1.5. The method of embodiment C1.3, wherein:-   each of the polynucleotide primer pairs hybridizes to the nucleic    acid of a pathogen that is different than the pathogens to which    each of the other polynucleotide primer pairs hybridize; and-   the presence, absence and/or amount of amplicons obtained from more    than one pathogen in (b) is determined in (c).-   C1.6. The method of any one of embodiments C1 to C1.5, wherein the    determining is by one or more of high-resolution melting (HRM),    quantitative PCR (qPCR), RT-PCR, quantitative RT-PCR (RT-qPCR),    loop-mediated isothermal amplification (LAMP), restriction    endonuclease digestion, gel electrophoresis and sequencing.-   C1.7. The method of any one of embodiments C1 to C1.6, wherein the    pathogen is a virus or viroid is selected from among Hops Latent    Viroid (HpLVd), Alfalfa Mosaic Virus (AMV), Beet Curly Top Virus    (BCTV), Hemp Streak Virus (HSV), Hemp Mosaic Virus (HMV), Tomato    spotted wilt virus (TSWV), Sunn-Hemp Mosaic Virus (SHMV), Arabis    Mosaic Virus (ArMV), Cucumber Mosaic Virus (CMV), Lettuce Chlorosis    Virus (LCV), Tobacco Ringspot Virus (TRSV), Tomato Ringspot Virus    (TomRSV), and Tobacco Streak Virus (TSV), Cannabis Cryptic Virus    (CCV), Potato Spindle Tubular Viroid (PSTV), Coconut cadang cadang    viroid (CCCV), Apple scar skin viroid (ASSV), Avocado sunblotch    viroid (ASBV), Tobacco streak virus (TSV), Tomato mosaic virus    (ToMV), Euonymous Ringspot Virus (ERSV), Elm Mosaic Virus (EMV), and    Hops Stunting Virus (HpSV).-   C1.8. A method for determining the presence, absence and/or amount    of a pathogen in a plant cultivar, comprising:-   (a) obtaining a nucleic acid sample from the plant cultivar;-   (b) contacting the nucleic acid sample with a polynucleotide primer    pair under amplification conditions and amplifying the sample,    thereby preparing an amplified nucleic acid mixture, wherein, if the    pathogen is present, the polynucleotide primer pair is capable of    specifically hybridizing to and amplifying a subsequence of the    nucleic acid of the pathogen, or to a complement thereof, wherein    the subsequence of the nucleic acid of the pathogen, or the    complement thereof, is non-identical to any subsequence of the    nucleic acid of the plant genome, or to any complement thereof; and-   (c) determining the presence, absence and/or amount of at least one    amplicon that is an amplification product of a polynucleotide primer    pair in the amplified nucleic acid mixture of (b) by qPCR or RT-qPCR    using more than one polynucleotide probe sequence, thereby    determining the presence, absence and/or amount of a pathogen in the    plant cultivar.-   C1.9. The method of embodiment C1.8, wherein the more than one    polynucleotide probe sequences hybridize to non-overlapping regions    of the subsequence of the pathogen that is amplified to generate the    amplicon.-   C1.10. The method of any one of embodiments C1 to C1.9, wherein the    pathogen is a virus or viroid is selected from among Hops Latent    Viroid (HpLVd), Alfalfa Mosaic Virus (AMV), Beet Curly Top Virus    (BCTV), Hemp Streak Virus (HSV), Hemp Mosaic Virus (HMV), Tomato    spotted wilt virus (TSWV), Sunn-Hemp Mosaic Virus (SHMV), Arabis    Mosaic Virus (ArMV), Cucumber Mosaic Virus (CMV), Lettuce Chlorosis    Virus (LCV), Tobacco Ringspot Virus (TRSV), Tomato Ringspot Virus    (TomRSV), and Tobacco Streak Virus (TSV), Cannabis Cryptic Virus    (CCV), Potato Spindle Tubular Viroid (PSTV), Coconut cadang cadang    viroid (CCCV), Apple scar skin viroid (ASSV), Avocado sunblotch    viroid (ASBV), Tobacco streak virus (TSV), Tomato mosaic virus    (ToMV), Euonymous Ringspot Virus (ERSV), Elm Mosaic Virus (EMV), and    Hops Stunting Virus (HpSV).-   C2. The method of any one of embodiments C1 to C1.10, wherein the    subsequence of the nucleic acid of the pathogen, or the complement    thereof, is in a region of overlap between two genes in the genome    of the pathogen.-   C3. The method of any one of embodiments C1 to C1.10 and C2, wherein    the pathogen is a virus or viroid.-   C4. The method of embodiment C3, wherein the virus or viroid    comprises nucleic acid that is DNA, or RNA, or DNA and RNA.-   C5. The method of embodiment C3 or embodiment C4, wherein the virus    or viroid is selected from among Hops Latent Viroid (HpLVd), Alfalfa    Mosaic Virus (AMV), Beet Curly Top Virus (BCTV), Hemp Streak Virus    (HSV), Hemp Mosaic Virus (HMV), Tomato spotted wilt virus (TSWV),    Sunn-Hemp Mosaic Virus (SHMV), Arabis Mosaic Virus (ArMV), Cucumber    Mosaic Virus (CMV), Lettuce Chlorosis Virus (LCV), Tobacco Ringspot    Virus (TRSV), Tomato Ringspot Virus (TomRSV), and Tobacco Streak    Virus (TSV), Cannabis Cryptic Virus (CCV), Potato Spindle Tubular    Viroid (PSTV), Coconut cadang cadang viroid (CCCV), Apple scar skin    viroid (ASSV), Avocado sunblotch viroid (ASBV), Tobacco streak virus    (TSV), Tomato mosaic virus (ToMV), Euonymous Ringspot Virus (ERSV),    Elm Mosaic Virus (EMV), and Hops Stunting Virus (HpSV).-   C6. The method of any one of embodiments C1 to C5, wherein the    subsequence of the nucleic acid of the pathogen, or the complement    thereof, comprises at least exon or at least one portion within an    exon.-   C7. The method of any one of embodiments C1 to C6, wherein the    subsequence comprises more than one exon or more than one portion    within an exon of at least two different genes.-   C8. The method of any one of embodiments C1 to C7, wherein the    subsequence of the nucleic acid of the pathogen, or the complement    thereof, comprises more than one exon or more than one portion    within an exon of at least two different genes.-   C9. The method of any one of embodiments C1 to C8, wherein the    method further comprises:    -   in (b), contacting the nucleic acid sample with at least one        second polynucleotide primer pair under amplification conditions        and amplifying the sample, thereby preparing an amplified        nucleic acid mixture, wherein the second polynucleotide primer        pair is capable of specifically hybridizing to and amplifying a        subsequence of the nucleic acid of the plant genome, or to a        complement thereof, wherein the subsequence of the nucleic acid        of the plant genome, or the complement thereof, is non-identical        to any subsequence of the nucleic acid of the pathogen, or to        any complement thereof; and    -   in (c), determining the presence, absence and/or amount of at        least one amplicon that is an amplification product of the        second polynucleotide primer pair, thereby determining whether        the amplification conditions are effective for generating        amplicons.-   C9.1. The method of any one of embodiments C1 to C9, wherein the    plant is of the subclass Rosidae.-   C10. The method of any one of embodiments C1 to C9.1, wherein the    plant is a Cannabis cultivar.-   C11. The method of embodiment C10, wherein the Cannabis cultivar is    selected from among Jamaican Lion, Purple Kush, CannaTsu, Finola,    Valley Fire and Cherry Chem.-   C12. The method of embodiment C10, wherein the plant genome is a    Cannabis sativa eudicots CS10 genome assembly.-   C12.1. The method of embodiment C10, wherein the Cannabis cultivar    is selected from among one or more of Type 1, Type 2, Type 3, Type 4    and Type 5 cultivars.-   C13. The method of any one of embodiments C9 to C12.1, wherein the    subsequence of the nucleic acid of the plant genome comprises all or    a portion of a gene that is conserved among species of the plant.-   C14. The method of any one of embodiments C9 to C13, wherein the    subsequence of the nucleic acid of the plant genome is of a    housekeeping gene or a portion thereof.-   C15. The method of embodiment C13 or C14, wherein the conserved gene    or housekeeping gene of the plant genome is selected from among 26S    rRNA, beta-tubulin, ATP Synthase, an rRNA subunit,    glyceraldehyde-3-phosphate dehydrogenase, Ubiquitin-conjugating    enzyme E2, eukaryotic transcription factors, eukaryotic initiation    factor 1 and beta-actin.-   C16. The method of any one of embodiments C1 to C15, wherein the    subsequence of the nucleic acid of the pathogen, or the complement    thereof, comprises all or a portion of at least one gene that is    conserved among species of that pathogen.-   C17. The method of embodiment C16, wherein the at least one gene    that is conserved among species of the pathogen is selected from    among RNA-3 coat protein, SS-ds-DNA Regulator protein, Movement    Protein, Pathogenesis Enhancer Protein, Rolling Circle Replication    Protein, Cell Cycle Regulator Protein and Replication Enhancer    Protein.-   C18. The method of any one of embodiments C1 to C17, wherein the    determining is by one or more of high-resolution melting (HRM),    quantitative PCR (qPCR), RT-PCR, quantitative RT-PCR (RT-qPCR),    loop-mediated isothermal amplification (LAMP), restriction    endonuclease digestion, gel electrophoresis and sequencing.-   C19. The method of embodiment C18, wherein the determining is by    qPCR or by RT-qPCR.-   C19.1 The method of embodiment C19, wherein the determining    comprises quantifying the at least one amplicon generated under    amplification conditions wherein the at least one polynucleotide    primer pair is substantially hybridized to and amplifies the    subsequence of the nucleic acid of the pathogen, or the complement    thereof, if present in the sample.-   C20. The method of any one of embodiments C1 to C19.1, wherein the    pathogen is Alfalfa Mosaic Virus (AMV).-   C21. The method of embodiment C20, wherein the subsequence of the    nucleic acid of the pathogen to which the polynucleotide primer pair    is capable of hybridizing comprises SEQ ID NO:91, or a portion of    SEQ ID NO:91, or a complement of SEQ ID NO:91, or a portion of the    complement of SEQ ID NO:91.-   C22. The method of embodiment C20 or C21, wherein the polynucleotide    primer pairs comprise: one primer selected from among those having    the sequences set forth in SEQ ID NOS: 80, 82 and 85, or from among    sequences that share 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,    99% or more identity with the sequences set forth in SEQ ID NOS: 80,    82 and 85; and one primer selected from among those having the    sequences set forth in SEQ ID NOS: 81, 83, 84 and 86; or from among    sequences that share 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,    99% or more identity with the sequences set forth in SEQ ID NOS: 81,    83, 84 and 86.-   C23. The method of any one of embodiments C20 to C22, wherein the at    least one amplicon generated under amplification conditions wherein    the at least one polynucleotide primer pair is substantially    hybridized to and amplifies the subsequence of the nucleic acid of    the pathogen, or the complement thereof, if present in the sample,    is quantified using a polynucleotide probe.-   C24. The method of embodiment C23, wherein the polynucleotide probe    is selected from among the sequences set forth in SEQ ID NOS: 87-90,    or from among sequences that share 90%, 91%, 92%, 93%, 94%, 95%,    96%, 97%, 98%, 99% or more identity with the sequences set forth in    SEQ ID NOS: 87-90.-   C25. The method of any one of embodiments C1 to C19.1, wherein the    pathogen is HpLVd.-   C26. The method of embodiment C25, wherein the subsequence of the    nucleic acid of the pathogen to which the polynucleotide primer pair    is capable of hybridizing comprises SEQ ID NO:1, or a portion of SEQ    ID NO:1, or a complement of SEQ ID NO:1, or a portion of the    complement of SEQ ID NO:1.-   C27. The method of embodiment C25 or C26, wherein one or more of the    polynucleotide primer pairs comprise:    -   (i) one thermomutant-specific primer selected from among those        having the sequences set forth in SEQ ID NOS: 2 and 77, or from        among sequences that share 90%, 91%, 92%, 93%, 94%, 95%, 96%,        97%, 98%, 99% or more identity with the sequences set forth in        SEQ ID NOS: 2 and 77; and one thermomutant-specific primer        selected from among those having the sequences set forth in SEQ        ID NOS: 7, 14, 15 and 78; or from among sequences that share        90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more        identity with the sequences set forth in SEQ ID NOS: 7, 14, 15        and 78; and/or    -   (ii) one thermomutant-resistant primer selected from among those        having the sequences set forth in SEQ ID NOS: 4, 6, 9, 11 and        13, or from among sequences that share 90%, 91%, 92%, 93%, 94%,        95%, 96%, 97%, 98%, 99% or more identity with the sequences set        forth in SEQ ID NOS: 4, 6, 9, 11 and 13; and one        thermomutant-resistant primer selected from among those having        the sequences set forth in SEQ ID NOS: 3, 5, 8, 10 and 12; or        from among sequences that share 90%, 91%, 92%, 93%, 94%, 95%,        96%, 97%, 98%, 99% or more identity with the sequences set forth        in SEQ ID NOS: 3, 5, 8, 10 and 12.-   C28. The method of any one of embodiments C25 to C27, wherein the at    least one amplicon generated under amplification conditions wherein    the at least one polynucleotide primer pair is substantially    hybridized to and amplifies the subsequence of the nucleic acid of    the pathogen, or the complement thereof, if present in the sample,    is quantified using a polynucleotide probe.-   C29. The method of embodiment C23, wherein the polynucleotide probe    is selected from among the sequences set forth in SEQ ID NOS: 16-20    and 79, or from among sequences that share 90%, 91%, 92%, 93%, 94%,    95%, 96%, 97%, 98%, 99% or more identity with the sequences set    forth in SEQ ID NOS: 16-20 and 79.-   C30. The method of any one of embodiments C1 to C19.1, wherein the    pathogen is BCTV.-   C31. The method of embodiment C30, wherein the subsequence of the    nucleic acid of the pathogen to which the polynucleotide primer pair    is capable of hybridizing is selected from among SEQ ID NOS:110,    112, 114, 116, 118 or 120, or a portion of SEQ ID NOS:110, 112, 114,    116, 118 or 120, or a complement of SEQ ID NOS:110, 112, 114, 116,    118 or 120, or a portion of the complement of SEQ ID NOS:110, 112,    114, 116, 118 or 120, or to regions of overlap that spans any two of    SEQ ID NOS:110, 112, 114, 116, 118 or 120 in the genome of the    pathogen.-   C32. The method of embodiment C31, wherein the subsequence of the    nucleic acid of the pathogen to which the polynucleotide primer pair    is capable of hybridizing is in a region of overlap that spans:    -   (i) the gene encoding the SS-ds-DNA Regulator Protein (SEQ ID        NO:110) and the gene encoding Movement Protein (SEQ ID NO:112);    -   (ii) the gene encoding the Pathogenesis Enhancement Protein (SEQ        ID NO:116) and the gene encoding the Rolling Circle Replication        Protein (SEQ ID NO:114);    -   (iii) the gene encoding the Rolling Circle Replication Protein        (SEQ ID NO:114) and the gene encoding the Cell Cycle Regulator        Protein (SEQ ID NO:118); or    -   (iv) the gene encoding the Pathogenesis Enhancement Protein (SEQ        ID NO:116) and the gene encoding the Replication Enhancer        Protein (SEQ ID NO:120).-   C33. The method of embodiment C32, wherein the polynucleotide primer    pairs comprise:    -   for (i), the primer pair having the sequences set forth in SEQ        ID NOS: 93 and 94 or sequences that share 90%, 91%, 92%, 93%,        94%, 95%, 96%, 97%, 98%, 99% or more identity with the sequences        set forth in SEQ ID NOS: 93 and 94, or the primer pair having        the sequences set forth in SEQ ID NOS: 93 and 105, or sequences        that share 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or        more identity with the sequences set forth in SEQ ID NOS: 93 and        105;    -   for (ii), the primer pair having the sequences set forth in SEQ        ID NOS: 96 and 97, or sequences that share 90%, 91%, 92%, 93%,        94%, 95%, 96%, 97%, 98%, 99% or more identity with the sequences        set forth in SEQ ID NOS: 96 and 97;    -   for (iii), the primer pair having the sequences set forth in SEQ        ID NOS: 99 and 100, or sequences that share 90%, 91%, 92%, 93%,        94%, 95%, 96%, 97%, 98%, 99% or more identity with the sequences        set forth in SEQ ID NOS: 99 and 100; and    -   for (iv), the primer pair having the sequences set forth in SEQ        ID NOS: 102 and 103, or sequences that share 90%, 91%, 92%, 93%,        94%, 95%, 96%, 97%, 98%, 99% or more identity with the sequences        set forth in SEQ ID NOS: 102 and 103.-   C34. The method of any one of embodiments C32 or C33, wherein the at    least one amplicon generated under amplification conditions wherein    the at least one polynucleotide primer pair is substantially    hybridized to and amplifies the subsequence of the nucleic acid of    the pathogen, or the complement thereof, if present in the sample,    is quantified using a polynucleotide probe.-   C35. The method of embodiment C34, wherein the polynucleotide probe    comprises:    -   for (i), the polynucleotide probe having the sequence set forth        in SEQ ID NO: 95 or a sequence that shares 90%, 91%, 92%, 93%,        94%, 95%, 96%, 97%, 98%, 99% or more identity with the sequence        set forth in SEQ ID NO: 95, and/or the polynucleotide probe        having the sequence set forth in SEQ ID NO: 106 or a sequence        that shares 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or        more identity with the sequence set forth in SEQ ID NO: 106;    -   for (ii), the polynucleotide probe having the sequence set forth        in SEQ ID NO: 98 or a sequence that shares 90%, 91%, 92%, 93%,        94%, 95%, 96%, 97%, 98%, 99% or more identity with the sequence        set forth in SEQ ID NO: 98;    -   for (iii), the polynucleotide probe having the sequence set        forth in SEQ ID NO: 101 or a sequence that shares 90%, 91%, 92%,        93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity with the        sequence set forth in SEQ ID NO:101; and    -   for (iv), the polynucleotide probe having the sequence set forth        in SEQ ID NO: 104 or a sequence that shares 90%, 91%, 92%, 93%,        94%, 95%, 96%, 97%, 98%, 99% or more identity with the sequence        set forth in SEQ ID NO: 104.-   C36. The method of any one of embodiments C30 to C35, wherein the    nucleic acid sample and/or the amplified nucleic acid mixture    comprises genomic DNA of the pathogen, if the pathogen is present in    the plant cultivar.-   C37. The method of any one of embodiments C30 to C35, wherein the    nucleic acid sample and/or the amplified nucleic acid mixture    comprises RNA or cDNA of the pathogen, if the pathogen is present in    the plant cultivar.-   C38. The method of any one of embodiments C1 to C19.1, wherein the    pathogen is a DNA virus or viroid and the nucleic acid sample and/or    the amplified nucleic acid mixture comprises genomic DNA of the    pathogen, if the pathogen is present in the plant cultivar.-   C39. The method of any one of embodiments C1 to C19.1, wherein the    pathogen is a DNA virus or viroid or an RNA virus or viroid and the    nucleic acid sample and/or the amplified nucleic acid mixture    comprises RNA or cDNA of the pathogen, if the pathogen is present in    the plant cultivar.-   C40. The method of any one of embodiments C1 to C39, wherein the    presence, absence and/or amount of a plurality of pathogens are    determined in the plant cultivar.-   C41. The method of embodiment C40, wherein the presence, absence    and/or amount of more than one of the plurality of pathogens is    determined simultaneously.-   C42. The method of embodiment C41, wherein the pathogens are viruses    and/or viroids.-   C43. The method of embodiment C42, wherein the presence and/or    amount of more than one virus and/or viroid is selected from among    Hops Latent Viroid (HpLVd), Alfalfa Mosaic Virus (AMV), Beet Curly    Top Virus (BCTV), Hemp Streak Virus (HSV), Hemp Mosaic Virus (HMV),    Tomato spotted wilt virus (TSWV), Sunn-Hemp Mosaic Virus (SHMV),    Arabis Mosaic Virus (ArMV), Cucumber Mosaic Virus (CMV), Lettuce    Chlorosis Virus (LCV), Tobacco Ringspot Virus (TRSV), Tomato    Ringspot Virus (TomRSV), Tobacco Streak Virus (TSV), Cannabis    Cryptic Virus (CCV), Potato Spindle Tubular Viroid (PSTV), Coconut    cadang cadang viroid (CCCV), Apple scar skin viroid (ASSV), Avocado    sunblotch viroid (ASBV), Tobacco streak virus (TSV), Tomato mosaic    virus (ToMV), Euonymous Ringspot Virus (ERSV), Elm Mosaic Virus    (EMV), and Hops Stunting Virus (HpSV), is determined simultaneously.-   C44. The method of embodiment C43, wherein the presence and/or    amount of more than one virus and/or viroid selected from among Hops    Latent Viroid (HpLVd), Alfalfa Mosaic Virus (AMV) and Beet Curly Top    Virus (BCTV) is determined simultaneously.-   C45. The method of any one of embodiments C1 to C44, wherein the    determining is by quantitative PCR (qPCR), or quantitative RT-PCR    (RT-qPCR), and an amplicon of at least one pathogen is quantified    with more than one polynucleotide probe sequence, wherein the    polynucleotide probe sequences hybridize to non-overlapping regions    of the subsequence of the pathogen that is amplified to generate the    amplicon.-   C45.1. The method of embodiment C45, further comprising, obtaining    the Cq value for each polynucleotide probe sequence.-   C46. The method of embodiment C45, wherein, if the Cq value obtained    with a first polynucleotide probe sequence is significantly    different than the Cq value obtained with any of the other    non-overlapping polynucleotide probe sequences, a variant in the    genotype of the pathogen is identified where the first    polynucleotide probe sequence binds to a subsequence of the pathogen    and, if the Cq values obtained with a first polynucleotide probe    sequence is similar to the Cq value obtained with any of the other    non-overlapping polynucleotide probe sequences, the genotype of the    pathogen is identified as not comprising a variant sequence where    the first polynucleotide probe sequence binds to a subsequence of    the pathogen.-   C47. The method of embodiment C46, wherein the presence or absence    of a variant in the genotype of the pathogen is correlated to the    infectivity of the pathogen.-   C48. The method of embodiment C46 or C47, wherein the presence or    absence of a variant in the genotype of the pathogen is correlated    to resistance or susceptibility of the plant to infection by the    pathogen comprising the genotype or a variant thereof.-   C48.0. The method of embodiment C48, wherein resistance or    susceptibility is measured by determining whether the plant is: (a)    infected and symptomatic when exposed to the pathogen or genotypic    variant thereof; (b) infected and asymptomatic when exposed to the    pathogen or genotypic variant thereof; or (c) resistant to infection    when exposed to the pathogen or genotypic variant thereof.-   C48.1. The method of embodiment C48, wherein, if the plant is    identified as resistant to infection by the pathogen or a genotypic    variant thereof, or asymptomatic, the plant is identified as    desirable for breeding, or as desirable for cultivating as a crop.-   C48.2. The method of embodiment C48.1, further comprising, breeding    the plant or cultivating the plant as a crop.-   C48.3. The method of any one of embodiments C48, C48.1 or C48.2,    wherein the plant is a Cannabis plant.-   C48.4. The method of any one of embodiments C48 to C48.3, wherein    the breeding produces at least one progeny plant that is resistant    to infection by a pathogen or genotypic variant thereof, or is    asymptomatic when infected by a pathogen or genotypic variant    thereof.-   C48.5. A method of removing symptomatic, infected plants from a    crop, comprising:    -   (a) identifying a plant as resistant, symptomatic or        asymptomatic when exposed to a pathogen by the method of        embodiment C48.0;    -   (b) selecting the plant for breeding one or more progeny plants        by the method of embodiment C48.1;    -   (c) breeding the plant to produce at least one progeny plant by        the method of embodiment C48.4; and    -   (d) replacing at least one symptomatic, infected plant in the        crop with at least one progeny plant that is resistant to        infection by a pathogen or genotypic variant thereof, or is        asymptomatic when infected by a pathogen or genotypic variant        thereof.-   C48.6. The method of embodiment C48.5, wherein (a), (b), (c) and (d)    are repeated until a majority or all of the symptomatic, infected    plants in the crop are replaced with progeny plants that are    resistant to infection by a pathogen or genotypic variant thereof,    or are asymptomatic when infected by a pathogen or genotypic variant    thereof-   C49. The method of any one of embodiments C1 to C48.6, comprising:    -   if the presence, absence and/or amount of one pathogen in the        plant cultivar is to be determined, obtaining more than one        amplicon by amplifying more than one subsequence of the nucleic        acid of the pathogen, or complements thereof, using more than        one polynucleotide primer pair, and determining the presence,        absence and/or amount of the pathogen by determining the        presence, absence and/or amount of at least two amplicons that        are 300 base pairs or less and are amplification products of the        more than one polynucleotide primer pair in the amplified        nucleic acid mixture of (b), thereby determining the presence,        absence and/or amount of a pathogen in the plant cultivar; or    -   if the presence, absence and/or amount of a plurality of        pathogens in the plant cultivar is to be determined, obtaining        more than one amplicon by amplifying more than one subsequence        of the nucleic acid of more than one of the plurality of        pathogens, or complements thereof, using more than one        polynucleotide primer pair for each of the more than one        pathogens, and determining the presence, absence and/or amount        of the more than one pathogens by determining the presence,        absence and/or amount of at least two amplicons for each        pathogen that are 300 base pairs or less and are amplification        products of the more than one polynucleotide primer pair in each        of the more than one pathogens of the amplified nucleic acid        mixture of (b), thereby determining the presence, absence and/or        amount of the more than one pathogens in the plant cultivar.-   C50. The method of embodiment C49, wherein, based on the presence    and/or relative amounts of the more than one amplicon, a variant in    the genotype of the pathogen(s) is/are identified or the genotype of    the pathogen(s) is/are identified as not comprising a variant    sequence.-   C51. The method of embodiment C49 or C50, wherein the presence or    absence of a variant in the genotype of the pathogen(s) is    correlated to resistance or susceptibility of the plant to infection    by the pathogen(s) comprising the genotype or a variant thereof.-   C51.1. The method of embodiment C51, wherein, if the plant is    identified as resistant to infection by the pathogen(s) or a    genotypic variant thereof, the plant is identified as desirable for    breeding, or as desirable for cultivating as a crop.-   C51.2. The method of embodiment C51.1, further comprising, breeding    the plant or cultivating the plant as a crop.-   C51.3. The method of any one of embodiments C51, C51.1 or C51.2,    wherein the plant is a Cannabis plant.-   C52. The method of any one of embodiments C49 to C51.3, wherein at    least one of the pathogens is a virus or a viroid.-   C52.1. The method of embodiment C52, wherein the virus or viroid is    selected from among Hops Latent Viroid (HpLVd), Alfalfa Mosaic Virus    (AMV), Beet Curly Top Virus (BCTV), Hemp Streak Virus (HSV), Hemp    Mosaic Virus (HMV), Tomato spotted wilt virus (TSWV), Sunn-Hemp    Mosaic Virus (SHMV), Arabis Mosaic Virus (ArMV), Cucumber Mosaic    Virus (CMV), Lettuce Chlorosis Virus (LCV), Tobacco Ringspot Virus    (TRSV), Tomato Ringspot Virus (TomRSV), and Tobacco Streak Virus    (TSV, Cannabis Cryptic Virus (CCV), Potato Spindle Tubular Viroid    (PSTV), Coconut cadang cadang viroid (CCCV), Apple scar skin viroid    (ASSV), Avocado sunblotch viroid (ASBV), Tobacco streak virus (TSV),    Tomato mosaic virus (ToMV), Euonymous Ringspot Virus (ERSV), Elm    Mosaic Virus (EMV), and Hops Stunting Virus (HpSV).-   C53. The method of embodiment C52 or C52.1, wherein the at least one    pathogen is a viroid, and the viroid is HpLVd.-   C54. The method of embodiment C53, wherein at least one amplicon is    obtained by amplifying a subsequence of the nucleic acid of the    pathogen that is thermomutant-resistant, and at least one amplicon    is obtained by amplifying a subsequence of the nucleic acid of the    pathogen that is thermomutant-specific.-   C54.1. The method of embodiment C54, wherein the polynucleotide    primer pairs for amplifying subsequence of the nucleic acid of the    pathogen that is thermomutant-resistant and the subsequence of the    nucleic acid of the pathogen that is thermomutant-specific are    selected from among:    -   (i) one thermomutant-specific primer selected from among those        having the sequences set forth in SEQ ID NOS: 2 and 77, or from        among sequences that share 90%, 91%, 92%, 93%, 94%, 95%, 96%,        97%, 98%, 99% or more identity with the sequences set forth in        SEQ ID NOS: 2 and 77; and one thermomutant-specific primer        selected from among those having the sequences set forth in SEQ        ID NOS: 7, 14, 15 and 78; or from among sequences that share        90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more        identity with the sequences set forth in SEQ ID NOS: 7, 14, 15        and 78; and/or    -   (ii) one thermomutant-resistant primer selected from among those        having the sequences set forth in SEQ ID NOS: 4, 6, 9, 11 and        13, or from among sequences that share 90%, 91%, 92%, 93%, 94%,        95%, 96%, 97%, 98%, 99% or more identity with the sequences set        forth in SEQ ID NOS: 4, 6, 9, 11 and 13; and one        thermomutant-resistant primer selected from among those having        the sequences set forth in SEQ ID NOS: 3, 5, 8, 10 and 12; or        from among sequences that share 90%, 91%, 92%, 93%, 94%, 95%,        96%, 97%, 98%, 99% or more identity with the sequences set forth        in SEQ ID NOS: 3, 5, 8, 10 and 12.-   C55. The method of embodiment C54 or C54.1, wherein, based on the    presence and/or relative amounts of the more than one amplicon, a    thermomutant variant in the genotype of the at least one pathogen is    identified, or the genotype of the at least one pathogen is    identified as not comprising a thermomutant variant sequence.-   C56. The method of embodiment C54, C54.1 or C55, wherein the    presence or absence of a thermomutant variant in the genotype of at    least one pathogen is correlated to resistance or susceptibility of    the plant to infection by the pathogen comprising the genotype or a    variant thereof.-   C57. The method of embodiment C52 or C52.1, wherein the at least one    pathogen is a virus, and the virus is AMV.-   C57.1. The method of embodiment C57, wherein the polynucleotide    primer pairs for amplifying the more than one amplicon are selected    from among:    -   one primer selected from among those having the sequences set        forth in SEQ ID NOS: 80, 82 and 85, or from among sequences that        share 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more        identity with the sequences set forth in SEQ ID NOS: 80, 82 and        85; and    -   one primer selected from among those having the sequences set        forth in SEQ ID NOS: 81, 83, 84 and 86; or from among sequences        that share 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or        more identity with the sequences set forth in SEQ ID NOS: 81,        83, 84 and 86.-   C58. The method of embodiment C52 or C52.1, wherein the at least one    pathogen is a virus, and the virus is BCTV.-   C58.1. The method of embodiment C58, wherein the polynucleotide    primer pairs for amplifying the more than one amplicon are selected    from among polynucleotide primer pairs capable of hybridizing to a    subsequence of the nucleic acid of the pathogen that is in a region    of overlap that spans:    -   (i) the gene encoding the SS-ds-DNA Regulator Protein (SEQ ID        NO:110) and the gene encoding Movement Protein (SEQ ID NO:112);    -   (ii) the gene encoding the Pathogenesis Enhancement Protein (SEQ        ID NO:116) and the gene encoding the Rolling Circle Replication        Protein (SEQ ID NO:114);    -   (iii) the gene encoding the Rolling Circle Replication Protein        (SEQ ID NO:114) and the gene encoding the Cell Cycle Regulator        Protein (SEQ ID NO:118); or    -   (iv) the gene encoding the Pathogenesis Enhancement Protein (SEQ        ID NO:116) and the gene encoding the Replication Enhancer        Protein (SEQ ID NO:120).-   C58.2. The method of embodiment C58.1, wherein the polynucleotide    primer pairs comprise:    -   for (i), the primer pair having the sequences set forth in SEQ        ID NOS: 93 and 94 or sequences that share 90%, 91%, 92%, 93%,        94%, 95%, 96%, 97%, 98%, 99% or more identity with the sequences        set forth in SEQ ID NOS: 93 and 94, or the primer pair having        the sequences set forth in SEQ ID NOS: 93 and 105, or sequences        that share 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or        more identity with the sequences set forth in SEQ ID NOS: 93 and        105;    -   for (ii), the primer pair having the sequences set forth in SEQ        ID NOS: 96 and 97, or sequences that share 90%, 91%, 92%, 93%,        94%, 95%, 96%, 97%, 98%, 99% or more identity with the sequences        set forth in SEQ ID NOS: 96 and 97;    -   for (iii), the primer pair having the sequences set forth in SEQ        ID NOS: 99 and 100, or sequences that share 90%, 91%, 92%, 93%,        94%, 95%, 96%, 97%, 98%, 99% or more identity with the sequences        set forth in SEQ ID NOS: 99 and 100; and    -   for (iv), the primer pair having the sequences set forth in SEQ        ID NOS: 102 and 103, or sequences that share 90%, 91%, 92%, 93%,        94%, 95%, 96%, 97%, 98%, 99% or more identity with the sequences        set forth in SEQ ID NOS: 102 and 103.-   C59. The method of any one of embodiments C1 to C58.2, wherein a    plurality of plant cultivars are analyzed for the presence and/or    amount of one or more pathogens.-   C60. The method of embodiment C59, wherein the plant cultivars are    of the same species.-   C61. The method of embodiment C59 or C60, wherein one or more plant    cultivars is/are a Cannabis cultivar.-   C62. The method of any one of embodiments C1 to C61, wherein a    plurality of Cannabis plant cultivars are analyzed.-   C63. The method of any one of embodiments C1 to C62, wherein the    size of the at least one amplicon that is amplified is 200 base    pairs or less.-   C64. The method of embodiment C63, wherein the size of the at least    one amplicon that is amplified is between about 40 base pairs to    about 200 base pairs.-   C65. The method of embodiment C64, wherein the size of the at least    one amplicon that is amplified is between about 50 base pairs to    about 150 base pairs.-   C66. The method of any one of embodiments C1 to C65, wherein    identification and/or quantitation of the amplicon is by a signal or    a label.-   C67. The method of embodiment C66, wherein the signal or label is    selected from among an electrical signal, an electronic signal, a    signal from an optical label or a radiolabel.-   C68. The method of embodiment C67, wherein the identification and/or    quantitation of the amplicon is by an optical label.-   C69. The method of embodiment C68, wherein the optical label is a    chromophore, a dye, or a fluorescent label.-   C70. The method of any one of embodiments C66 to C69, wherein:    -   a plurality of amplicons are analyzed using a plurality of        polynucleotide primer pairs, and/or    -   a plurality of polynucleotide probes are used to quantitate an        amplicon, and    -   the plurality of amplicons and/or the plurality of        polynucleotide probes are each associated with a unique signal        or label for identification and/or quantitation.-   C71. The method of any one of embodiments C1 to C70, wherein the    nucleic acid sample from the plant cultivar is on a solid support    and (b) and (c) are performed on the solid support.-   C72. The method of embodiment C71, wherein the presence, absence    and/or amount of more than one pathogen in the plant cultivar is    determined.-   C73. The method of embodiment C71 or C72, wherein the presence,    absence and/or amount of one or more pathogens in a plurality of    plant cultivars is determined.-   C74. The method of any one of embodiments C71 to C73, wherein at    least one plant cultivar is Cannabis.-   C75. The method of embodiment C73, wherein all of the plurality of    plant cultivars are Cannabis cultivars.-   C76. The method of any one of embodiments C1 to C75 that is    performed on a FTA® card.-   D1. A method of preparing a polynucleotide primer pair for    specifically hybridizing to and amplifying nucleic acid of a plant    pathogen, comprising:    -   (a) Identifying a polynucleotide primer pair that is capable of        specifically hybridizing to and amplifying a polynucleotide        comprising a subsequence of the nucleic acid of a plant        pathogen, or a complement thereof, wherein the plant is capable        of being infected by the pathogen and the subsequence of the        nucleic acid of the pathogen, or the complement thereof, is        non-identical to any subsequence of the nucleic acid of the        plant genome, or to any complement thereof;    -   (b) identifying whether the subsequence of the nucleic acid of        the pathogen is conserved among species of the pathogen; and    -   (c) if the subsequence of the nucleic acid of the pathogen is        conserved among species of the pathogen, preparing the        polynucleotide primer pair.-   D1.1. The method of embodiment D1, wherein the subsequence of the    nucleic acid of the pathogen, or the complement thereof, comprises    at least one exon of at least one gene of the pathogen.-   D2. The method of embodiment D1, wherein the size of the product    that is amplified by the prepared polynucleotide primer pair is 300    base pairs or less.-   D3. The method of embodiment D1 or D2, wherein the size of the    product that is amplified by the prepared polynucleotide primer pair    is 200 base pairs or less.-   D4. The method of any one of embodiments D1 to D3, wherein the size    of the product that is amplified by the prepared polynucleotide    primer pair is between about 40 base pairs to about 200 base pairs.-   D5. The method of any one of embodiments D1 to D4, wherein the size    of the product that is amplified by the prepared polynucleotide    primer pair is between about 50 base pairs to about 150 base pairs.-   D6. The method of any one of embodiments D1 to D5, wherein the    melting temperature of each primer hybridized to its target    conserved sequence is between about 57° C. to about 63° C.-   D7. The method of any one of embodiments D1 to D6, wherein the    difference between the melting temperatures of each primer of the    primer pair hybridized to its target sequence is 3° C. or less.-   D8. The method of any one of embodiments D1 to D7, wherein the    subsequence of the nucleic acid of the pathogen, or the complement    thereof, comprises more than one exon of at least one gene of the    pathogen.-   D9. The method of any one of embodiments D1 to D8, wherein the    subsequence of the nucleic acid of the pathogen, or the complement    thereof, comprises more than one exon spanning more than one gene of    the pathogen.-   D10. The method of any one of embodiments D1 to D9, further    comprising, preparing at least one polynucleotide probe for    quantifying the product that is amplified by the prepared    polynucleotide pair.-   D11. The method of embodiment D10, comprising preparing more than    one polynucleotide probe for quantifying the product that is    amplified by the prepared polynucleotide pair, wherein each    polynucleotide probe binds to a subsequence that does not overlap    with the subsequences to which the other polynucleotide probes bind.-   D12. The method of any one of embodiments D1 to D11, wherein more    than one polynucleotide primer pair is prepared, and each    polynucleotide primer pair binds to a subsequence that does not    overlap with the subsequences to which the other polynucleotide    primer pairs bind.-   D13. The method of any one of embodiments D1 to D12, wherein the    pathogen is a virus or viroid.-   D14. The method of embodiment D13, wherein the virus or viroid is    selected from among Hops Latent Viroid (HpLVd), Alfalfa Mosaic Virus    (AMV), Beet Curly Top Virus (BCTV), Hemp Streak Virus (HSV), Hemp    Mosaic Virus (HMV), Tomato spotted wilt virus (TSWV), Sunn-Hemp    Mosaic Virus (SHMV), Arabis Mosaic Virus (ArMV), Cucumber Mosaic    Virus (CMV), Lettuce Chlorosis Virus (LCV), Tobacco Ringspot Virus    (TRSV), Tomato Ringspot Virus (TomRSV), Tobacco Streak Virus (TSV),    Cannabis Cryptic Virus (CCV), Potato Spindle Tubular Viroid (PSTV),    Coconut cadang cadang viroid (CCCV), Apple scar skin viroid (ASSV),    Avocado sunblotch viroid (ASBV), Tobacco streak virus (TSV), Tomato    mosaic virus (ToMV), Euonymous Ringspot Virus (ERSV), Elm Mosaic    Virus (EMV), and Hops Stunting Virus (HpSV).-   D15. The method of embodiment D14, wherein the virus or viroid is    selected from among Hops Latent Viroid (HpLVd), Alfalfa Mosaic Virus    (AMV) and Beet Curly Top Virus (BCTV).-   E1. A composition, comprising one or more polynucleotide primer    pairs prepared by the method of any one of embodiments D1 to D15    and, optionally, one or more polynucleotide probes prepared by the    method of any one of embodiments D10 to D15.-   E1.1. A composition, comprising one or more polynucleotide primer    pairs used in the method of any one of embodiments C1 to C70 for    specifically hybridizing to and amplifying nucleic acid of a plant    pathogen and, optionally, one or more polynucleotide probes for    quantifying one or more amplicons generated using the one or more    polynucleotide primer pairs.-   E1.2. The composition of embodiment E1 or E1.1, wherein the pathogen    is a virus or viroid.-   E1.3. The composition of embodiment E1.2, wherein the virus or    viroid is selected from among Hops Latent Viroid (HpLVd), Alfalfa    Mosaic Virus (AMV), Beet Curly Top Virus (BCTV), Hemp Streak Virus    (HSV), Hemp Mosaic Virus (HMV), Tomato spotted wilt virus (TSWV),    Sunn-Hemp Mosaic Virus (SHMV), Arabis Mosaic Virus (ArMV), Cucumber    Mosaic Virus (CMV), Lettuce Chlorosis Virus (LCV), Tobacco Ringspot    Virus (TRSV), Tomato Ringspot Virus (TomRSV), Tobacco Streak Virus    (TSV), Cannabis Cryptic Virus (CCV), Potato Spindle Tubular Viroid    (PSTV), Coconut cadang cadang viroid (CCCV), Apple scar skin viroid    (ASSV), Avocado sunblotch viroid (ASBV), Tobacco streak virus (TSV),    Tomato mosaic virus (ToMV), Euonymous Ringspot Virus (ERSV), Elm    Mosaic Virus (EMV), and Hops Stunting Virus (HpSV).-   E2. The composition of any one of embodiments E1 to E1.3, wherein at    least one polynucleotide primer pair is capable of specifically    hybridizing to and amplifying a subsequence of the nucleic acid of    Alfalfa Mosaic Virus (AMV).-   E3. The composition of embodiment E2, wherein the subsequence of the    nucleic acid of the Alfalfa Mosaic Virus (AMV) to which the    polynucleotide primer pair is capable of hybridizing comprises SEQ    ID NO:91, or a portion of SEQ ID NO:91, or a complement of SEQ ID    NO:91, or a portion of the complement of SEQ ID NO:91.-   E4. The composition of embodiment E2 or E3, wherein the at least one    polynucleotide primer pair is selected from among: one primer    selected from among those having the sequences set forth in SEQ ID    NOS: 80, 82 and 85, or from among sequences that share 90%, 91%,    92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity with the    sequences set forth in SEQ ID NOS: 80, 82 and 85; and one primer    selected from among those having the sequences set forth in SEQ ID    NOS: 81, 83, 84 and 86; or from among sequences that share 90%, 91%,    92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity with the    sequences set forth in SEQ ID NOS: 81, 83, 84 and 86.-   E5. The composition of any one of embodiments E1 to E4, further    comprising a polynucleotide probe.-   E6. The composition of embodiment E5, wherein the polynucleotide    probe is selected from among the sequences set forth in SEQ ID NOS:    87-90, or from among sequences that share 90%, 91%, 92%, 93%, 94%,    95%, 96%, 97%, 98%, 99% or more identity with the sequences set    forth in SEQ ID NOS: 87-90.-   E7. The composition of any one of embodiments E1 to E1.3, wherein at    least one polynucleotide primer pair is capable of specifically    hybridizing to and amplifying a subsequence of the nucleic acid of    HpLVd.-   E8. The composition of embodiment E7, wherein the subsequence of the    nucleic acid of the pathogen to which the at least one    polynucleotide primer pair is capable of hybridizing comprises SEQ    ID NO:1, or a portion of SEQ ID NO:1, or a complement of SEQ ID    NO:1, or a portion of the complement of SEQ ID NO:1.-   E9. The composition of embodiment E7 or E8, wherein the at least one    polynucleotide primer pair is selected from among:    -   (i) one thermomutant-specific primer selected from among those        having the sequences set forth in SEQ ID NOS: 2 and 77, or from        among sequences that share 90%, 91%, 92%, 93%, 94%, 95%, 96%,        97%, 98%, 99% or more identity with the sequences set forth in        SEQ ID NOS: 2 and 77; and one thermomutant-specific primer        selected from among those having the sequences set forth in SEQ        ID NOS: 7, 14, 15 and 78; or from among sequences that share        90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more        identity with the sequences set forth in SEQ ID NOS: 7, 14, 15        and 78; and/or    -   (ii) one thermomutant-resistant primer selected from among those        having the sequences set forth in SEQ ID NOS: 4, 6, 9, 11 and        13, or from among sequences that share 90%, 91%, 92%, 93%, 94%,        95%, 96%, 97%, 98%, 99% or more identity with the sequences set        forth in SEQ ID NOS: 4, 6, 9, 11 and 13; and one        thermomutant-resistant primer selected from among those having        the sequences set forth in SEQ ID NOS: 3, 5, 8, 10 and 12; or        from among sequences that share 90%, 91%, 92%, 93%, 94%, 95%,        96%, 97%, 98%, 99% or more identity with the sequences set forth        in SEQ ID NOS: 3, 5, 8, 10 and 12.-   E10. The composition of any one of embodiments E7 to E9, further    comprising a polynucleotide probe.-   E11. The composition of embodiment E10, wherein the polynucleotide    probe is selected from among the sequences set forth in SEQ ID NOS:    16-20 and 79, or from among sequences that share 90%, 91%, 92%, 93%,    94%, 95%, 96%, 97%, 98%, 99% or more identity with the sequences set    forth in SEQ ID NOS: 16-20 and 79.-   E12. The composition of any one of embodiments E1 to E1.3, wherein    at least one polynucleotide primer pair is capable of specifically    hybridizing to and amplifying a subsequence of the nucleic acid of    BCTV.-   E13. The composition of embodiment E12, wherein the subsequence of    the nucleic acid of the pathogen to which the at least one    polynucleotide primer pair is capable of hybridizing is selected    from among SEQ ID NOS:110, 112, 114, 116, 118 or 120, or a portion    of SEQ ID NOS:110, 112, 114, 116, 118 or 120, or a complement of SEQ    ID NOS:110, 112, 114, 116, 118 or 120, or a portion of the    complement of SEQ ID NOS:110, 112, 114, 116, 118 or 120, or to    regions of overlap that spans any two of SEQ ID NOS:110, 112, 114,    116, 118 or 120 in the genome of the pathogen.-   E14. The composition of embodiment E12 or E13, wherein the    subsequence of the nucleic acid of the pathogen to which the at    least one polynucleotide primer pair is capable of hybridizing is in    a region of overlap that spans:    -   (i) the gene encoding the SS-ds-DNA Regulator Protein (SEQ ID        NO:110) and the gene encoding Movement Protein (SEQ ID NO:112);    -   (ii) the gene encoding the Pathogenesis Enhancement Protein (SEQ        ID NO:116) and the gene encoding the Rolling Circle Replication        Protein (SEQ ID NO:114);    -   (iii) the gene encoding the Rolling Circle Replication Protein        (SEQ ID NO:114) and the gene encoding the Cell Cycle Regulator        Protein (SEQ ID NO:118); or    -   (iv) the gene encoding the Pathogenesis Enhancement Protein (SEQ        ID NO:116) and the gene encoding the Replication Enhancer        Protein (SEQ ID NO:120).-   E15. The composition of embodiment E14, wherein the polynucleotide    primer pairs comprise:    -   for (i), the primer pair having the sequences set forth in SEQ        ID NOS: 93 and 94 or sequences that share 90%, 91%, 92%, 93%,        94%, 95%, 96%, 97%, 98%, 99% or more identity with the sequences        set forth in SEQ ID NOS: 93 and 94, or the primer pair having        the sequences set forth in SEQ ID NOS: 93 and 105, or sequences        that share 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or        more identity with the sequences set forth in SEQ ID NOS: 93 and        105;    -   for (ii), the primers having the sequences set forth in SEQ ID        NOS: 96 and 97, or sequences that share 90%, 91%, 92%, 93%, 94%,        95%, 96%, 97%, 98%, 99% or more identity with the sequences set        forth in SEQ ID NOS: 96 and 97;    -   for (iii), the primers having the sequences set forth in SEQ ID        NOS: 99 and 100, or sequences that share 90%, 91%, 92%, 93%,        94%, 95%, 96%, 97%, 98%, 99% or more identity with the sequences        set forth in SEQ ID NOS: 99 and 100; and    -   for (iv), the primers having the sequences set forth in SEQ ID        NOS: 102 and 103, or sequences that share 90%, 91%, 92%, 93%,        94%, 95%, 96%, 97%, 98%, 99% or more identity with the sequences        set forth in SEQ ID NOS: 102 and 103.-   E16. The composition of any one of embodiments E12 to E15, further    comprising a polynucleotide probe.-   E17. The composition of embodiment E16, wherein the polynucleotide    probe comprises:    -   for (i), the polynucleotide probe having the sequence set forth        in SEQ ID NO: 95 or a sequence that shares 90%, 91%, 92%, 93%,        94%, 95%, 96%, 97%, 98%, 99% or more identity with the sequence        set forth in SEQ ID NO: 95, and/or the polynucleotide probe        having the sequence set forth in SEQ ID NO: 106 or a sequence        that shares 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or        more identity with the sequence set forth in SEQ ID NO: 106;    -   for (ii), the polynucleotide probe having the sequence set forth        in SEQ ID NO: 98 or a sequence that shares 90%, 91%, 92%, 93%,        94%, 95%, 96%, 97%, 98%, 99% or more identity with the sequence        set forth in SEQ ID NO: 98;    -   for (iii), the polynucleotide probe having the sequence set        forth in SEQ ID NO: 101 or a sequence that shares 90%, 91%, 92%,        93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity with the        sequence set forth in SEQ ID NO:101; and    -   for (iv), the polynucleotide probe having the sequence set forth        in SEQ ID NO: 104 or a sequence that shares 90%, 91%, 92%, 93%,        94%, 95%, 96%, 97%, 98%, 99% or more identity with the sequence        set forth in SEQ ID NO: 104.-   E18. The composition of any one of embodiments E1 to E17, further    comprising, a polynucleotide primer pair that is capable of    specifically hybridizing to and amplifying a subsequence of the    nucleic acid of the plant genome.-   E19. The composition of embodiment E18, wherein the subsequence of    the nucleic acid of the plant genome comprises all or a portion of a    gene that is conserved among species of the plant.-   E20. The composition of embodiment E18 or E19, wherein the    subsequence of the nucleic acid of the plant genome is of a    housekeeping gene or a portion thereof.-   E21. The composition of embodiment E19 or E20, wherein the conserved    gene or housekeeping gene of the plant genome is selected from among    26S rRNA, beta-tubulin, ATP Synthase, an rRNA subunit,    glyceraldehyde-3-phosphate dehydrogenase, Ubiquitin-conjugating    enzyme E2, eukaryotic transcription factors, eukaryotic initiation    factor 1 and beta-actin.-   F1. A kit, comprising one or more of the compositions of any one of    embodiments E1 to E21, and instructions for use.-   F2. The kit of embodiment F1, further comprising, at least one    signal or label.-   F3. The kit of embodiment F2, wherein the signal or label is    selected from among an electrical signal, an electronic signal, a    signal from an optical label or a radiolabel.-   F4. The kit of embodiment F3, comprising an optical label.-   F5. The kit of embodiment F4, wherein the optical label is a    chromophore, a dye, or a fluorescent label.-   G1. A solid support, comprising:    -   single-stranded nucleic acid from a plant cultivar; and    -   one or more polynucleotide primer pairs used in the method of        any one of embodiments C1 to C70 or prepared by the method of        any one of embodiments D1 to D15 for specifically hybridizing to        and amplifying nucleic acid of a plant pathogen.-   G2. The solid support of embodiment G1, wherein the single-stranded    nucleic acid from the plant cultivar is DNA, RNA or cDNA.-   G2.1. The solid support of embodiment G2, wherein the    single-stranded nucleic acid from the plant cultivar is DNA that    comprises genomic DNA.-   G3. The solid support of embodiment G1 or G2, wherein the pathogen    is a virus or viroid.-   G4. The solid support of embodiment G3, wherein the virus or viroid    is selected from among Hops Latent Viroid (HpLVd), Alfalfa Mosaic    Virus (AMV), Beet Curly Top Virus (BCTV), Hemp Streak Virus (HSV),    Hemp Mosaic Virus (HMV), Tomato spotted wilt virus (TSWV), Sunn-Hemp    Mosaic Virus (SHMV), Arabis Mosaic Virus (ArMV), Cucumber Mosaic    Virus (CMV), Lettuce Chlorosis Virus (LCV), Tobacco Ringspot Virus    (TRSV), Tomato Ringspot Virus (TomRSV), Tobacco Streak Virus (TSV),    Cannabis Cryptic Virus (CCV), Potato Spindle Tubular Viroid (PSTV),    Coconut cadang cadang viroid (CCCV), Apple scar skin viroid (ASSV),    Avocado sunblotch viroid (ASBV), Tobacco streak virus (TSV), Tomato    mosaic virus (ToMV), Euonymous Ringspot Virus (ERSV), Elm Mosaic    Virus (EMV), and Hops Stunting Virus (HpSV).-   G5. The solid support of any one of embodiments G1 to G4, comprising    more than one polynucleotide primer pair, wherein the polynucleotide    primer pairs specifically hybridize to non-overlapping subsequences    of the same pathogen, or the polynucleotide primer pairs    specifically hybridize to subsequences of different pathogens, or    some polynucleotide primer pairs specifically hybridize to    non-overlapping subsequences of the same pathogen and some    polynucleotide primer pairs specifically hybridize to subsequences    of different pathogens.-   G6. The solid support of any one of embodiments G1 to G5, wherein at    least one polynucleotide primer pair is capable of specifically    hybridizing to and amplifying a subsequence of the nucleic acid of    Alfalfa Mosaic Virus (AMV).-   G7. The solid support of embodiment G6, wherein the subsequence of    the nucleic acid of the Alfalfa Mosaic Virus (AMV) to which the    polynucleotide primer pair is capable of hybridizing comprises SEQ    ID NO:91, or a portion of SEQ ID NO:91, or a complement of SEQ ID    NO:91, or a portion of the complement of SEQ ID NO:91.-   G8. The solid support of embodiment G6 or G7, wherein the at least    one polynucleotide primer pair is selected from among: one primer    selected from among those having the sequences set forth in SEQ ID    NOS: 80, 82 and 85, or from among sequences that share 90%, 91%,    92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity with the    sequences set forth in SEQ ID NOS: 80, 82 and 85; and one primer    selected from among those having the sequences set forth in SEQ ID    NOS: 81, 83, 84 and 86; or from among sequences that share 90%, 91%,    92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity with the    sequences set forth in SEQ ID NOS: 81, 83, 84 and 86.-   G9. The solid support of any one of embodiments G1 to G8, further    comprising a polynucleotide probe.-   G10. The solid support of embodiment G9, wherein the polynucleotide    probe is selected from among the sequences set forth in SEQ ID NOS:    87-90, or from among sequences that share 90%, 91%, 92%, 93%, 94%,    95%, 96%, 97%, 98%, 99% or more identity with the sequences set    forth in SEQ ID NOS: 87-90.-   G11. The solid support of any one of embodiments G1 to G5, wherein    at least one polynucleotide primer pair is capable of specifically    hybridizing to and amplifying a subsequence of the nucleic acid of    HpLVd.-   G12. The solid support of embodiment G11, wherein the subsequence of    the nucleic acid of the pathogen to which the at least one    polynucleotide primer pair is capable of hybridizing comprises SEQ    ID NO:1, or a portion of SEQ ID NO:1, or a complement of SEQ ID    NO:1, or a portion of the complement of SEQ ID NO:1.-   G13. The solid support of embodiment G11 or G12, wherein the at    least one polynucleotide primer pair is selected from among:    -   (i) one thermomutant-specific primer selected from among those        having the sequences set forth in SEQ ID NOS: 2 and 77, or from        among sequences that share 90%, 91%, 92%, 93%, 94%, 95%, 96%,        97%, 98%, 99% or more identity with the sequences set forth in        SEQ ID NOS: 2 and 77; and one thermomutant-specific primer        selected from among those having the sequences set forth in SEQ        ID NOS: 7, 14, 15 and 78; or from among sequences that share        90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more        identity with the sequences set forth in SEQ ID NOS: 7, 14, 15        and 78; and/or    -   (ii) one thermomutant-resistant primer selected from among those        having the sequences set forth in SEQ ID NOS: 4, 6, 9, 11 and        13, or from among sequences that share 90%, 91%, 92%, 93%, 94%,        95%, 96%, 97%, 98%, 99% or more identity with the sequences set        forth in SEQ ID NOS: 4, 6, 9, 11 and 13; and one        thermomutant-resistant primer selected from among those having        the sequences set forth in SEQ ID NOS: 3, 5, 8, 10 and 12; or        from among sequences that share 90%, 91%, 92%, 93%, 94%, 95%,        96%, 97%, 98%, 99% or more identity with the sequences set forth        in SEQ ID NOS: 3, 5, 8, 10 and 12.-   G14. The solid support of any one of embodiments G11 to G13, further    comprising a polynucleotide probe.-   G15. The solid support of embodiment G14, wherein the polynucleotide    probe is selected from among the sequences set forth in SEQ ID NOS:    16-20 and 79, or from among sequences that share 90%, 91%, 92%, 93%,    94%, 95%, 96%, 97%, 98%, 99% or more identity with the sequences set    forth in SEQ ID NOS: 16-20 and 79.-   G16. The solid support of any one of embodiments G1 to G5, wherein    at least one polynucleotide primer pair is capable of specifically    hybridizing to and amplifying a subsequence of the nucleic acid of    BCTV.-   G17. The solid support of embodiment G16, wherein the subsequence of    the nucleic acid of the pathogen to which the at least one    polynucleotide primer pair is capable of hybridizing is selected    from among SEQ ID NOS:110, 112, 114, 116, 118 or 120, or a portion    of SEQ ID NOS:110, 112, 114, 116, 118 or 120, or a complement of SEQ    ID NOS:110, 112, 114, 116, 118 or 120, or a portion of the    complement of SEQ ID NOS:110, 112, 114, 116, 118 or 120, or to    regions of overlap that spans any two of SEQ ID NOS:110, 112, 114,    116, 118 or 120 in the genome of the pathogen.-   G18. The solid support of embodiment G16 or G17, wherein the    subsequence of the nucleic acid of the pathogen to which the at    least one polynucleotide primer pair is capable of hybridizing is in    a region of overlap that spans:    -   (i) the gene encoding the SS-ds-DNA Regulator Protein (SEQ ID        NO:110) and the gene encoding Movement Protein (SEQ ID NO:112);    -   (ii) the gene encoding the Pathogenesis Enhancement Protein (SEQ        ID NO:116) and the gene encoding the Rolling Circle Replication        Protein (SEQ ID NO:114);    -   (iii) the gene encoding the Rolling Circle Replication Protein        (SEQ ID NO:114) and the gene encoding the Cell Cycle Regulator        Protein (SEQ ID NO:118); or    -   (iv) the gene encoding the Pathogenesis Enhancement Protein (SEQ        ID NO:116) and the gene encoding the Replication Enhancer        Protein (SEQ ID NO:120).-   G19. The solid support of embodiment G18, wherein the polynucleotide    primer pairs comprise:    -   for (i), the primer pair having the sequences set forth in SEQ        ID NOS: 93 and 94 or sequences that share 90%, 91%, 92%, 93%,        94%, 95%, 96%, 97%, 98%, 99% or more identity with the sequences        set forth in SEQ ID NOS: 93 and 94, or the primer pair having        the sequences set forth in SEQ ID NOS: 93 and 105, or sequences        that share 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or        more identity with the sequences set forth in SEQ ID NOS: 93 and        105;    -   for (ii), the primers having the sequences set forth in SEQ ID        NOS: 96 and 97, or sequences that share 90%, 91%, 92%, 93%, 94%,        95%, 96%, 97%, 98%, 99% or more identity with the sequences set        forth in SEQ ID NOS: 96 and 97;    -   for (iii), the primers having the sequences set forth in SEQ ID        NOS: 99 and 100, or sequences that share 90%, 91%, 92%, 93%,        94%, 95%, 96%, 97%, 98%, 99% or more identity with the sequences        set forth in SEQ ID NOS: 99 and 100; and    -   for (iv), the primers having the sequences set forth in SEQ ID        NOS: 102 and 103, or sequences that share 90%, 91%, 92%, 93%,        94%, 95%, 96%, 97%, 98%, 99% or more identity with the sequences        set forth in SEQ ID NOS: 102 and 103.-   G20. The solid support of any one of embodiments G16 to G19, further    comprising a polynucleotide probe.-   G21. The solid support of embodiment G20, wherein the polynucleotide    probe comprises:    -   for (i), the polynucleotide probe having the sequence set forth        in SEQ ID NO: 95 or a sequence that shares 90%, 91%, 92%, 93%,        94%, 95%, 96%, 97%, 98%, 99% or more identity with the sequence        set forth in SEQ ID NO: 95, and/or the polynucleotide probe        having the sequence set forth in SEQ ID NO: 106 or a sequence        that shares 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or        more identity with the sequence set forth in SEQ ID NO: 106;    -   for (ii), the polynucleotide probe having the sequence set forth        in SEQ ID NO: 98 or a sequence that shares 90%, 91%, 92%, 93%,        94%, 95%, 96%, 97%, 98%, 99% or more identity with the sequence        set forth in SEQ ID NO: 98;    -   for (iii), the polynucleotide probe having the sequence set        forth in SEQ ID NO: 101 or a sequence that shares 90%, 91%, 92%,        93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity with the        sequence set forth in SEQ ID NO:101; and    -   for (iv), the polynucleotide probe having the sequence set forth        in SEQ ID NO: 104 or a sequence that shares 90%, 91%, 92%, 93%,        94%, 95%, 96%, 97%, 98%, 99% or more identity with the sequence        set forth in SEQ ID NO: 104.-   G22. The solid support of any one of embodiments G1 to G21, further    comprising, a polynucleotide primer pair that is capable of    specifically hybridizing to and amplifying a subsequence of the    nucleic acid of the plant genome.-   G23. The solid support of embodiment G22, wherein the subsequence of    the nucleic acid of the plant genome comprises all or a portion of a    gene that is conserved among species of the plant.-   G24. The solid support of embodiment G22 or G23, wherein the    subsequence of the nucleic acid of the plant genome is of a    housekeeping gene or a portion thereof.-   G25. The solid support of embodiment G23 or G24, wherein the    conserved gene or housekeeping gene of the plant genome is selected    from among 26S rRNA, beta-tubulin, ATP Synthase, an rRNA subunit,    glyceraldehyde-3-phosphate dehydrogenase, Ubiquitin-conjugating    enzyme E2, eukaryotic transcription factors, eukaryotic initiation    factor 1 and beta-actin.-   G26. The solid support of any one of embodiments G1 to G25 that    comprises a bead, column, capillary, disk, filter, dipstick,    membrane, wafer, comb, pin or a chip.-   G27. The solid support of any one of embodiments G1 to G26 that    comprises a material selected from among silicon, silica, glass,    controlled-pore glass (CPG), nylon, Wang resin, Merrifield resin,    Sephadex, Sepharose, cellulose, magnetic beads, Dynabeads, a metal,    a metal surface, a plastic or polymer or combinations thereof.-   G28. The solid support of any one of embodiments G1 to G27,    comprising a plurality of plant cultivars.-   G29. A collection of solid supports comprising two or more solid    supports of any one of embodiments G1 to G27, wherein each solid    support in the collection comprises nucleic acid from a different    plant cultivar.-   C29.1. The solid support of embodiment G28 or the collection of    embodiment G29, wherein at least one of the plant cultivars is of    the subclass Rosidae.-   G30. The solid support of embodiment G28 or the collection of    embodiment G29 or G29.1, wherein at least one of the plant cultivars    is a Cannabis cultivar.-   G31. The solid support of embodiment G28 or the collection of    embodiment G29, wherein more than one of the plant cultivars is a    Cannabis cultivar.-   G32. The solid support of embodiment G28 or the collection of    embodiment G29, wherein the plurality of plant cultivars are    Cannabis cultivars.

The entirety of each patent, patent application, publication anddocument referenced herein is incorporated by reference. Citation ofpatents, patent applications, publications and documents is not anadmission that any of the foregoing is pertinent prior art, nor does itconstitute any admission as to the contents or date of thesepublications or documents. Their citation is not an indication of asearch for relevant disclosures. All statements regarding the date(s) orcontents of the documents is based on available information and is notan admission as to their accuracy or correctness.

The technology has been described with reference to specificimplementations. The terms and expressions that have been utilizedherein to describe the technology are descriptive and not necessarilylimiting. Certain modifications made to the disclosed implementationscan be considered within the scope of the technology. Certain aspects ofthe disclosed implementations suitably may be practiced in the presenceor absence of certain elements not specifically disclosed herein.

Each of the terms “comprising,” “consisting essentially of,” and“consisting of” may be replaced with either of the other two terms. Theterm “a” or “an” can refer to one of or a plurality of the elements itmodifies (e.g., “a reagent” can mean one or more reagents) unless it iscontextually clear either one of the elements or more than one of theelements is described. The term “about” as used herein refers to a valuewithin 10% of the underlying parameter (i.e., plus or minus 10%; e.g., aweight of “about 100 grams” can include a weight between 90 grams and110 grams). Use of the term “about” at the beginning of a listing ofvalues modifies each of the values (e.g., “about 1, 2 and 3” refers to“about 1, about 2 and about 3”). When a listing of values is described,the listing includes all intermediate values and all fractional valuesthereof (e.g., the listing of values “80%, 85% or 90%” includes theintermediate value 86% and the fractional value 86.4%). When a listingof values is followed by the term “or more,” the term “or more” appliesto each of the values listed (e.g., the listing of “80%, 90%, 95%, ormore” or “80%, 90%, 95% or more” or “80%, 90%, or 95% or more” refers to“80% or more, 90% or more, or 95% or more”). When a listing of values isdescribed, the listing includes all ranges between any two of the valueslisted (e.g., the listing of “80%, 90% or 95%” includes ranges of “80%to 90%,” “80% to 95%” and “90% to 95%”).

Thus, it should be understood that although the present technology hasbeen specifically disclosed by representative embodiments and optionalfeatures, modification and variation of the concepts herein disclosedmay be resorted to by those skilled in the art, and such modificationsand variations are considered within the scope of this technology.

Certain embodiments of the technology are set forth in the claim(s) thatfollow(s).

What is claimed is:
 1. A method for generating nucleic acidamplification products from a plant sample, comprising: contacting thenucleic acid of the plant sample with a set of loop mediated isothermalamplification (LAMP) polynucleotide primers under the amplificationconditions, thereby generating one or more amplification products,wherein: the majority or all of the polynucleotide primers hybridize tosubsequences of SEQ ID NO:1, if present in the nucleic acid of the plantsample under the amplification conditions; the subsequences of SEQ IDNO:1 to which the majority or all of the polynucleotide primershybridize under the amplification conditions contain no variantnucleotide position; and each subsequence of SEQ ID NO:1 between thesubsequences to which the polynucleotide primers hybridize contain oneor more variant nucleotide positions.
 2. The method of claim 1,comprising analyzing the amplification products.
 3. The method of claim1, wherein the plant has been heat treated.
 4. The method of claim 1,wherein the plant has not been heat treated.
 5. The method of claim 1,wherein the plant is of the subclass Rosidae.
 6. The method of claim 5,wherein the plant is a Cannabis plant.
 7. The method of claim 1, whereineach polynucleotide in each primer pair comprises a sequence that isnon-identical to any subsequence, or complement thereof, in a Cannabisgenome.
 8. The method of claim 2, wherein the analyzing comprisesdetecting the presence or absence of a Hops Latent Viroid in the plant.9. The method of claim 8, wherein the analyzing comprises detecting oneor more genetic variations in a Hops Latent Viroid.
 10. The method ofclaim 1, wherein the LAMP polynucleotide primer set is chosen from oneor more of: a) a primer set comprising the polynucleotides of SEQ IDNO:21 to SEQ ID NO:29, b) a primer set comprising the polynucleotides ofSEQ ID NO:30 to SEQ ID NO:38, c) a primer set comprising thepolynucleotides of SEQ ID NO:39 to SEQ ID NO:47, and d) a primer setcomprising the polynucleotides of SEQ ID NO:48 to SEQ ID NO:56.
 11. Themethod of claim 2, wherein the analyzing comprises performing a highresolution melting (HRM) endpoint assay on the amplification products.12. The method of claim 11, wherein the analyzing comprises detectingone or more genetic variations in a Hops Latent Viroid according toresults obtained from the high resolution melting (HRM) endpoint assay.13. The method of claim 1, wherein the subsequences of SEQ ID NO:1 towhich the majority or all of the polynucleotide primers hybridize underthe amplification conditions contain no thermomutant positions.
 14. Themethod of claim 13, wherein the thermomutant positions are chosen fromone or more of nucleotide position 7 of SEQ ID NO:1, nucleotide position10 of SEQ ID NO:1, nucleotide position 12 of SEQ ID NO:1, nucleotideposition 26 of SEQ ID NO:1, nucleotide position 27 of SEQ ID NO:1,nucleotide position 28 of SEQ ID NO:1, nucleotide position 29 of SEQ IDNO:1, nucleotide position 30 of SEQ ID NO:1, nucleotide position 33 ofSEQ ID NO:1, nucleotide position 35 of SEQ ID NO:1, nucleotide position43 of SEQ ID NO:1, nucleotide position 59 of SEQ ID NO:1, nucleotideposition 121 of SEQ ID NO:1, nucleotide position 128 of SEQ ID NO:1,nucleotide position 134 of SEQ ID NO:1, nucleotide position 150 of SEQID NO:1, nucleotide position 157 of SEQ ID NO:1, nucleotide position 162of SEQ ID NO:1, nucleotide position 168 of SEQ ID NO:1, nucleotideposition 169 of SEQ ID NO:1, nucleotide position 177 of SEQ ID NO:1,nucleotide position 200 of SEQ ID NO:1, nucleotide position 225 of SEQID NO:1, nucleotide position 229 of SEQ ID NO:1, nucleotide position 247of SEQ ID NO:1, nucleotide position 248 of SEQ ID NO:1, and nucleotideposition 253 of SEQ ID NO:1.
 15. A composition, comprising a set of loopmediated isothermal amplification (LAMP) polynucleotide primers,wherein: the majority or all of the polynucleotide primers hybridize tosubsequences of SEQ ID NO:1, if present in the nucleic acid of the plantsample under the amplification conditions; the subsequences of SEQ IDNO:1 to which the majority or all of the polynucleotide primershybridize under the amplification conditions contain no variantnucleotide position; and each subsequence of SEQ ID NO:1 between thesubsequences to which the polynucleotide primers hybridize contain oneor more variant nucleotide positions.
 16. The composition of claim 15,wherein the LAMP polynucleotide primer set is chosen from one or moreof: a) a primer set comprising the polynucleotides of SEQ ID NO:21 toSEQ ID NO:29, b) a primer set comprising the polynucleotides of SEQ IDNO:30 to SEQ ID NO:38, c) a primer set comprising the polynucleotides ofSEQ ID NO:39 to SEQ ID NO:47, and d) a primer set comprising thepolynucleotides of SEQ ID NO:48 to SEQ ID NO:56.
 17. The composition ofclaim 15, wherein the subsequences of SEQ ID NO:1 to which the majorityor all of the polynucleotide primers hybridize contain no thermomutantpositions.
 18. The composition of claim 17, wherein the thermomutantpositions are chosen from one or more of nucleotide position 7 of SEQ IDNO:1, nucleotide position 10 of SEQ ID NO:1, nucleotide position 12 ofSEQ ID NO:1, nucleotide position 26 of SEQ ID NO:1, nucleotide position27 of SEQ ID NO:1, nucleotide position 28 of SEQ ID NO:1, nucleotideposition 29 of SEQ ID NO:1, nucleotide position 30 of SEQ ID NO:1,nucleotide position 33 of SEQ ID NO:1, nucleotide position 35 of SEQ IDNO:1, nucleotide position 43 of SEQ ID NO:1, nucleotide position 59 ofSEQ ID NO:1, nucleotide position 121 of SEQ ID NO:1, nucleotide position128 of SEQ ID NO:1, nucleotide position 134 of SEQ ID NO:1, nucleotideposition 150 of SEQ ID NO:1, nucleotide position 157 of SEQ ID NO:1,nucleotide position 162 of SEQ ID NO:1, nucleotide position 168 of SEQID NO:1, nucleotide position 169 of SEQ ID NO:1, nucleotide position 177of SEQ ID NO:1, nucleotide position 200 of SEQ ID NO:1, nucleotideposition 225 of SEQ ID NO:1, nucleotide position 229 of SEQ ID NO:1,nucleotide position 247 of SEQ ID NO:1, nucleotide position 248 of SEQID NO:1, and nucleotide position 253 of SEQ ID NO:1.
 19. The compositionof claim 15, comprising one or more further polynucleotide primerswherein: each polynucleotide of the one or more further polynucleotideprimers is identical, or substantially identical, to a subsequence ofSEQ ID NO:1, or complement thereof; each subsequence of SEQ ID NO:1, orcomplement thereof, to which each polynucleotide is identical, orsubstantially identical, contains one or more variant nucleotidepositions.
 20. The composition of claim 19, wherein each furtherpolynucleotide primer comprises a sequence that is non-identical to anysubsequence, or complement thereof, in a Cannabis genome.
 21. Thecomposition of claim 19, wherein the one or more further polynucleotideprimers independently are chosen from a polynucleotide comprising thesequence CTACGTGACTTACCTGTATGGTGGC (SEQ ID NO:2), GTGAAGAAGGAGCCGTTCCA(SEQ ID NO:7), AGAGTTGTATTCACCGGGTAGTTTCC (SEQ ID NO:14), orGCACTTTTTATGTGAACTTCTGC (SEQ ID NO:15).
 22. The composition of claim 21,wherein the one or more further polynucleotide primers consist ofCTACGTGACTTACCTGTATGGTGGC (SEQ ID NO:2), GTGAAGAAGGAGCCGTTCCA (SEQ IDNO:7), AGAGTTGTATTCACCGGGTAGTTTCC (SEQ ID NO:14), andGCACTTTTTATGTGAACTTCTGC (SEQ ID NO:15).
 23. A method for determining thepresence, absence and/or amount of at least one pathogen in a plantcultivar, comprising: (a) obtaining a nucleic acid sample from the plantcultivar; (b) contacting the nucleic acid sample with more than onepolynucleotide primer pair under amplification conditions and amplifyingthe sample, thereby preparing an amplified nucleic acid mixture,wherein, if at least one pathogen is present, at least onepolynucleotide primer pair is capable of specifically hybridizing to andamplifying a subsequence of the nucleic acid of the pathogen, or to acomplement thereof, wherein the subsequence of the nucleic acid of thepathogen, or the complement thereof, is non-identical to any subsequenceof the nucleic acid of the plant genome, or to any complement thereof;and (c) determining the presence, absence and/or amount of at least oneamplicon that is an amplification product of a polynucleotide primerpair in the amplified nucleic acid mixture of (b), thereby determiningthe presence, absence and/or amount of a pathogen in the plant cultivar.24. The method of claim 23, wherein: each of the polynucleotide primerpairs hybridizes to the nucleic acid of the same pathogen; eachpolynucleotide primer pair hybridizes to a subsequence of the nucleicacid of the pathogen that does not overlap with the subsequences towhich each of the other primer pairs hybridizes; and the presence,absence and/or amount of more than one amplicon of the pathogen that isobtained in (b) is determined in (c).
 25. The method of claim 23,wherein: each of the polynucleotide primer pairs hybridizes to thenucleic acid of a pathogen that is different than the pathogens to whicheach of the other polynucleotide primer pairs hybridize; and thepresence, absence and/or amount of amplicons obtained from more than onepathogen in (b) is determined in (c).
 26. The method of claim 23,wherein the determining is by one or more of high-resolution melting(HRM), quantitative PCR (qPCR), RT-PCR, quantitative RT-PCR (RT-qPCR),loop-mediated isothermal amplification (LAMP), restriction endonucleasedigestion, gel electrophoresis and sequencing.
 27. The method of claim23, wherein the pathogen is a virus or viroid is selected from amongHops Latent Viroid (HpLVd), Alfalfa Mosaic Virus (AMV), Beet Curly TopVirus (BCTV), Hemp Streak Virus (HSV), Hemp Mosaic Virus (HMV), Tomatospotted wilt virus (TSWV), Sunn-Hemp Mosaic Virus (SHMV), Arabis MosaicVirus (ArMV), Cucumber Mosaic Virus (CMV), Lettuce Chlorosis Virus(LCV), Tobacco Ringspot Virus (TRSV), Tomato Ringspot Virus (TomRSV),and Tobacco Streak Virus (TSV), Cannabis Cryptic Virus (CCV), PotatoSpindle Tubular Viroid (PSTV), Coconut cadang cadang viroid (CCCV),Apple scar skin viroid (ASSV), Avocado sunblotch viroid (ASBV), Tobaccostreak virus (TSV), Tomato mosaic virus (ToMV), Euonymous Ringspot Virus(ERSV), Elm Mosaic Virus (EMV), and Hops Stunting Virus (HpSV).
 28. Amethod for determining the presence, absence and/or amount of a pathogenin a plant cultivar, comprising: (a) obtaining a nucleic acid samplefrom the plant cultivar; (b) contacting the nucleic acid sample with apolynucleotide primer pair under amplification conditions and amplifyingthe sample, thereby preparing an amplified nucleic acid mixture,wherein, if the pathogen is present, the polynucleotide primer pair iscapable of specifically hybridizing to and amplifying a subsequence ofthe nucleic acid of the pathogen, or to a complement thereof, whereinthe subsequence of the nucleic acid of the pathogen, or the complementthereof, is non-identical to any subsequence of the nucleic acid of theplant genome, or to any complement thereof; and (c) determining thepresence, absence and/or amount of at least one amplicon that is anamplification product of a polynucleotide primer pair in the amplifiednucleic acid mixture of (b) by qPCR or RT-qPCR using more than onepolynucleotide probe sequence, thereby determining the presence, absenceand/or amount of a pathogen in the plant cultivar.
 29. The method ofclaim 28, wherein the more than one polynucleotide probe sequenceshybridize to non-overlapping regions of the subsequence of the pathogenthat is amplified to generate the amplicon.
 30. The method of claim 28,wherein the pathogen is a virus or viroid is selected from among HopsLatent Viroid (HpLVd), Alfalfa Mosaic Virus (AMV), Beet Curly Top Virus(BCTV), Hemp Streak Virus (HSV), Hemp Mosaic Virus (HMV), Tomato spottedwilt virus (TSWV), Sunn-Hemp Mosaic Virus (SHMV), Arabis Mosaic Virus(ArMV), Cucumber Mosaic Virus (CMV), Lettuce Chlorosis Virus (LCV),Tobacco Ringspot Virus (TRSV), Tomato Ringspot Virus (TomRSV), andTobacco Streak Virus (TSV), Cannabis Cryptic Virus (CCV), Potato SpindleTubular Viroid (PSTV), Coconut cadang cadang viroid (CCCV), Apple scarskin viroid (ASSV), Avocado sunblotch viroid (ASBV), Tobacco streakvirus (TSV), Tomato mosaic virus (ToMV), Euonymous Ringspot Virus(ERSV), Elm Mosaic Virus (EMV), and Hops Stunting Virus (HpSV).